Cells producing fc containing molecules having altered glycosylation patterns and methods and use thereof

ABSTRACT

The present application relates to the field of glyco-engineering, more specifically to glyco-engineering of Fc-containing molecules, such as antibodies. It is shown herein that Fc-containing molecules with a specific glycosylation pattern have a considerably longer circulating half-life in vivo, without having an altered binding affinity for their respective antigen. This has therapeutic implications in reducing the frequency with which these molecules need to be administered, without affecting therapeutic efficacy. Also, cells are provided that can produce the Fc molecules with the desired glycosylation pattern.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claims priority to U.S.application Ser. No. 14/917,248, filed Mar. 7, 2016, which claimspriority to a national phase entry under 35 U.S.C. § 371 ofInternational Patent Application PCT/EP2014/068946, filed Sep. 5, 2014,designating the United States of America and published in English asInternational Patent Publication WO 2015/032899 A1 on Mar. 12, 2015,which claims the benefit under Article 8 of the Patent CooperationTreaty to European Patent Application Serial No. 13183124.0, filed Sep.5, 2013.

TECHNICAL FIELD

This application relates to the field of glyco-engineering, morespecifically to glyco-engineering of Fc-containing molecules, such asantibodies. It is shown herein that Fc-containing molecules with aspecific glycosylation pattern have a considerably longer circulatinghalf-life in vivo, without having an altered binding affinity for theirrespective antigen. This has therapeutic implications in reducing thefrequency with which these molecules need to be administered, withoutaffecting therapeutic efficacy. Also, cells are provided that canproduce the Fc molecules with the desired glycosylation pattern.

BACKGROUND

Antibodies, and particularly IgG antibodies, are the basis of some ofthe most successful therapeutics developed over the last 20 years (e.g.,bevacizumab, rituximab, infliximab, adalimumab, trastuzumab, orcetuximab, to name but a few). This success is at least in partattributable to the fact that they are highly specific, have long serumhalf-lives, and can be produced relatively routinely, making them idealdrugs for immunotherapy. The basic structure of an antibody molecule (orimmunoglobulin, Ig) is comprised of two identical heavy and twoidentical light polypeptide chains. These chains are linked by disulfidebonds forming a “Y”-shaped structure. Human immunoglobulins can becategorized into five classes (IgG, IgA, IgD, IgE, and IgM) referencingthe heavy chain. IgG and IgA antibodies are further separated into four(IgG1-4) and two subclasses (IgA1-2), respectively. Recognition ofspecific antigens is mediated by the antigen-binding fragment (Fab),which includes the variable regions and one constant domain of the lightand heavy chains. Effector functions are initiated by binding of thefragment-crystallizable region (Fc), corresponding to the other twodomains of the constant region of the heavy chain (CH2 and CH3), toeffector proteins such as Fc receptors (FcRs). Thus, the Fab fragmentsare comprised of variable and constant domains of light and heavychains, while Fc fragments are comprised entirely of constant domains ofheavy chains. This Fc domain prolongs the serum half-life of antibodiesdue to pH-dependent binding to the neonatal Fc receptor (FcRn), whichsalvages the protein from being degraded in endosomes.

Given the long serum half-life of antibodies, construction of Fc-fusionproteins has been implemented to prolong the half-life of therapeuticproteins, as most biologically active proteins and peptides have veryshort serum half-lives due to fast renal clearance, which limits theirexposure in the target tissue and, consequently, their pharmacologicaleffects. The Fc-fusion strategy also met with considerable success:marketed Fc-fusion proteins include, e.g., etanercept, alefacept,abatacept, rilonacept, romiplostim, belatacept, and aflibercept. As anadditional benefit, the Fc portion of Fc-fusion proteins allows easierexpression and protein A-affinity purification, which confers practicaladvantages in the development of antibody and Fc-fusion therapeutics.

Antibody engineering approaches have been used to further advance theclinical success of therapeutic antibodies, e.g., by altering theirbinding properties to ligand or Fc receptors, or by further extendingtheir half-life. Typical approaches to achieve this include introducingmutations or altering glycosylation of the antibodies. Introducingmutations in the Fc chain has the inherent drawback of no longer workingwith natural sequences. Contrary to glycosylation of therapeuticproteins, which is generally accepted to prolong circulating half-life,studies on the effect of glycosylation on the elimination rate ofimmunoglobulins from circulation have produced conflicting results(Millward et al., 2008), and most studies conclude that glycanstructural differences of the Fc moiety do not affect clearance (Chen etal., 2009).

During post-translational modification of the antibody chains, enzymesin the endoplasmic reticulum and Golgi apparatus can attach carbohydratechains to the polypeptide backbone of the antibody. A single N-linkedglycan is present in the Fc portion of all IgG subclasses, at anasparagine at position 297 (Kabat numbering). About 20% of IgGantibodies contain glycans elsewhere on the molecule (Jefferis, 2005).Most recombinant antibody drugs have been engineered or selected tocontain only the single Fc glycosylation site.

When the antibody chains are correctly folded and associated, theoligosaccharide at position 297 is sequestered within an internal spaceenclosed by the CH2 domains, and there are extensive non-covalentinteractions between the oligosaccharide and the amino acids ofantibody, resulting in reciprocal influences on conformation.

The oligosaccharides found at the conserved Asn-297 site are typicallyof a fucosylated biantennary complex type. However, among antibodymolecules, there may be considerable heterogeneity in the carbohydratestructures (glycoforms) due to altered branching, chain length and/oraltered number of carbohydrate moieties. Indeed, the structure of theattached N-linked oligosaccharides varies considerably, depending on thedegree of processing, and can include high-mannose, as well as complexbiantennary oligosaccharides with or without bisecting GlcNAc and coreFucose residues (Wright and Morrison, 1997). Typically, there isheterogeneous processing of the core oligosaccharide structures attachedat a given glycosylation site, with the result that even monoclonalantibodies exist as multiple glycoforms. Moreover, major differences inantibody glycosylation occur between antibody-producing cell lines, andeven minor differences are seen for a given cell line grown underdifferent culture conditions.

Indeed, each step in mammalian N-glycan biosynthesis (FIG. 1A, top) is<100% efficient, and some enzymes compete for substrates, resulting inmany different glycoforms. Heterogeneous glycosylation presents problemsin the production of therapeutic proteins. For example, glycans canaffect pharmacokinetics and biological activities (Ferrara et al., 2006;Elliott et al., 2004); however, N-glycans are often crucial for proteinfolding, so these difficulties cannot be overcome by completely removingN-glycosylation sites or interfering with glycosylation before or in theendoplasmic reticulum.

The differences in glycoforms may result in different or inconsistenteffector functions, which can render the antibodies difficult to usetherapeutically or define from a regulatory point of view. Also,glycoforms that are not commonly biosynthesized in humans may beallergenic, immunogenic and accelerate the plasmatic clearance of thelinked antibody. Deglycosylating the Fc moiety at position 297 canresult in decreased or eliminated effector functions of theFc-containing molecules, or in reduced stability (Krapp et al., 2003;Yamaguchi et al., 2006; Barb et al., 2011; Buck et al., 2013).

It would be advantageous to obtain Fc-containing molecules that haveimproved properties, such as longer circulating half-life, but withoutdrawbacks such as heterogeneous glycosylation or reduced antigenbinding.

BRIEF SUMMARY

Provided are ways of producing antibodies and Fc-fusion proteins thathave a prolonged half-life in circulation. Also provided are antibodiesand Fc-fusion proteins with a much less heterogeneous glycosylationprofile than obtained in normal mammalian cells.

Upon establishing an animal cell line that was glyco-engineered to yieldglycoproteins with very specific simple glycans, it was surprisinglyfound that Fc-containing molecules produced in this cell line have amuch longer circulation time in vivo. As the antibodies were otherwiseidentical to that produced in non-glyco-engineered cells, the differenceis solely attributable to the specific glycosylation pattern.

Accordingly, in a first aspect, cells are provided that contain:

-   -   a first exogenous nucleic acid sequence encoding an        endoglucosaminidase enzyme;    -   a second exogenous nucleic acid sequence encoding an        Fc-containing molecule.

It is particularly envisaged that the cells are higher eukaryotic cells.According to further specific embodiments, the higher eukaryotic cellsare vertebrate cells, in particular, mammalian cells. Examples include,but are not limited to, CHO cells or HEK293 cells (e.g., HEK293S cells).According to particular embodiments, the Fc part of the Fc-containingmolecule is an Fc of an IgG-type molecule.

According to particular embodiments, the glycosyltransferase GnTI,encoded by the gene MGAT1, is inactivated in the cells.

According to specific embodiments, the expression of theendoglucosaminidase enzyme is targeted to the Golgi apparatus. This canbe achieved, e.g., by operably linking the endoglucosaminidase to aGolgi localization signal.

According to particular embodiments, the endoglucosaminidase enzyme is amannosyl-glycoprotein endo-beta-N-acetylglucosaminidase (E.C. 3.2.1.96).Different such enzymes exist, e.g., Endo T, Endo H, Endo S, ENGase. Aparticularly envisaged enzyme is Endo T.

According to a further aspect, Fc-containing molecules are provided thatare obtainable by producing them in these cells, i.e., Fc-containingmolecules produced in higher eukaryotic cells characterized by thepresence of:

-   -   a first exogenous nucleic acid sequence encoding an        endoglucosaminidase enzyme;    -   a second exogenous nucleic acid sequence encoding the        Fc-containing molecule.

Producing Fc-containing molecules in these cells will lead to moleculeswith a specific glycosylation pattern. Accordingly, Fc-containingmolecules are provided, characterized in that the glycosylation on theFc part consists of a glycan selected from the following: atrisaccharide structure Neu5Ac-Hex-HexNAc, a disaccharide structureHex-HexNAc, and a monosaccharide structure HexNAc. According to veryspecific embodiments, the glycan is selected from the trisaccharidestructure and the disaccharide structure (i.e., is not a structureexisting of a single HexNAc, such as a single GlcNAc).

Most particularly, the glycosylation on the Fc part is glycosylation onresidue N297 of the Fc part. This is a conserved residue in the Fcmoiety of IgG-like molecules.

As Fc molecules with a single glycosylation site typically have oneglycan chain only, also provided is a plurality of identicalFc-containing molecules, characterized in that the glycosylation (e.g.,the glycosylation on N297) of the Fc part consists of one or moreglycans selected from the following: a trisaccharide structureNeu5Ac-Hex-HexNAc, a disaccharide structure Hex-HexNAc, and amonosaccharide structure HexNAc. According to particular embodiments, atleast one of the plurality of Fc-containing molecules has a glycanselected from the trisaccharide structure and the disaccharidestructure; i.e., at least one of the plurality of Fc-containingmolecules has a glycan that is not a monosaccharide structure HexNAc.

According to further particular embodiments, the glycans on theFc-containing molecule or of the plurality of Fc-containing moleculesare selected from the trisaccharide structureNeu5Ac-α-2,3-Gal-β-1,4-GlcNAc, the disaccharide structureGal-β-1,4-GlcNAc, and the monosaccharide structure GlcNAc.

According to particular embodiments, the Fc-containing molecule with thespecific glycosylation is an antibody, in particular an IgG.

According to a further aspect, the Fc-containing molecules describedherein are provided for use as a medicament. For instance, theFc-containing molecules may be provided for use in intravenousimmunoglobulin therapy. This is equivalent as saying that methods oftreating a subject with intravenous immunoglobulin therapy are provided,comprising administering to the subject an Fc-containing moleculeproduced by the cells described herein. Or alternatively, methods oftreating a subj ect with intravenous immunoglobulin therapy areprovided, comprising administering to the subj ect an Fc-containingmolecule (or a plurality of Fc-containing molecules), characterized inthat the glycosylation on the Fc part consists of a glycan selected fromthe following: a trisaccharide structure Neu5Ac-Hex-HexNAc, adisaccharide structure Hex-HexNAc, and a monosaccharide structureHexNAc.

According to yet further aspects, methods for producing Fc-containingmolecules with a specific glycosylation pattern on residue N297 in ahigher eukaryotic cell are provided, comprising the steps of:

-   -   providing a higher eukaryotic cell comprising a first exogenous        nucleic acid sequence encoding an endoglucosaminidase enzyme,        wherein the endoglucosaminidase is operably linked to a Golgi        localization signal, and a second exogenous nucleic acid        sequence encoding the Fc-containing molecule, in conditions        suitable for expressing the endoglucosaminidase enzyme and the        Fc-containing molecule; and    -   recovering the Fc-containing molecule after it has been        intracellularly contacted with the endoglucosaminidase.

According to specific embodiments, the Fc-containing molecules that areproduced are secreted.

It is a particular advantage that the Fc-containing molecules withspecific glycosylation patterns have a longer circulating half-life;i.e., they remain longer in circulation, are cleared less efficiently,or maintain a certain threshold concentration for a longer period oftime than Fc-containing molecules that don't have an alteredglycosylation pattern. This is in fact surprising, since it is generallyassumed that complex glycosylation is beneficial in prolongingcirculating half-life. Moreover, the Fc-containing molecules (e.g.,antibodies) do not only remain longer in circulation, but the affinityof these antibodies for their ligands is not affected by the alteredglycosylation pattern.

Thus, Fc-containing molecules with altered glycosylation patterns asdescribed herein are provided, which retain antigen binding activity andhave increased circulation time in vivo compared to non-modifiedglycoforms. In these embodiments, the Fc-containing molecules areFc-containing molecules that bind antigen. For instance, theFc-containing molecule can be an antibody, but can also be a chimericFc-fusion protein, wherein the Fc moiety is fused to a binding moiety(e.g., a nanobody, a Fab, a F(ab′)₂).

Accordingly, methods are provided to increase circulation time of anFc-containing molecule to be administered to a subject in need thereof,without altering antigen binding, comprising:

-   -   providing an Fc-containing molecule as described herein;    -   administering the Fc-containing molecule to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1D: The GlycoDelete strategy and cell line characterization.Sugar residues: blue square, N-acetylglucosamine; green circle, mannose;yellow circle, galactose; purple diamond, sialic acid. FIG. 1A: Inmammalian cells with intact glycosylation machinery (top), oligomannoseglycans entering the Golgi are further trimmed by class I mannosidases(ManI) to Man₅GlcNAc₂ forms. They are committed to hybrid or complextype N-glycans, upon modification by N-acetylglucosaminyltransferase 1(GnTI) with a β-1,2-N-acetylglucosamine on the α-1,3-mannose. Multipleglycosylhydrolases and glycosyltransferases further model complex typeN-glycans through many biosynthetic steps (WT glycosylation, blackarrows in top), leading to substantial heterogeneity. In the 293SGnTI−/−line, glycans are committed to the oligomannose type. These N-glycansare hydrolyzed by Golgi-targeted endoT in GlycoDelete cells, resultingin single N-acetylglucosamine residues (GlycoDelete glycoengineering,bottom). The single GlcNAc stumps can be elongated by galactosyl- andsialyltransferases (GalT and SiaT) in the Golgi. pHopt, pH optimum. FIG.1B: The concanavalin A selection strategy directly selects for thedesired glycan phenotype, as full deglycosylation of cell surfaceglycoproteins by endoT would result in the absence of ConA ligands,rendering cells resistant against conA. The parental GnTI−/− cells diewhen treated with conA. FIG. 1C: Growth curve for 293SGnTI−/− and 293SGlycoDelete cells counted every 24 hours. Error bars represent SDs foreach triplicate (Table 4). Both lines show comparable growth kinetics.FIG. 1D: Scatterplot of average (n=3) gene expression values of 7,344genes for 293 SGlycoDelete versus 293 SGnTI(−) cells. The correlationcoefficient is 0.9865. Significantly differentially expressed genes(moderated t-test in which the standard errors have been moderatedacross genes according to a simple Bayesian model; P<0.01) are labeledwith their names. Microarray signal intensities <8 on the representedscale were too low for reliable detection.

FIG. 2: Evaluation of the two different trans Golgi targeting domains(GM₂S-endoT and ST-endoT) compared to secreted endoT (s-endoT). In thisexperiment, two trans Golgi targeting sequences were evaluated as towhich is most effective at retaining a fusion of these sequences withthe endoT catalytic domain inside 293 SGnTI−/− cells. For comparison, asecreted version of endoT (i.e., with a secretion signal but no Golgitargeting sequences) was analyzed. The Western blots of SDS-PAGEseparated cell lysate proteins and of proteins present in the cellcultivation medium were developed with a polyclonal anti-endoT antiserumor with a monoclonal anti-c-Myc epitope antibody. The c-Myc epitope isC-terminally fused to the different protein constructs and its presenceor absence thus came to the conclusion on C-terminal processing of theproteins. From these results, it is clear that the GM₂S-derived sequenceis ineffective at retaining endoT intracellularly, as this constructyields the same distribution of intra- and extracellular endoT forms asthe secreted version of the protein. It appears that the GM₂ sequence isefficiently cleaved off. To the contrary, the ST-derived sequenceeffectively retains endoT intracellularly and the major band at 50 kDamatches the expected molecular mass of the ST-endoT fusion protein. Someminor secretion of two C-terminally proteolyzed forms still occurs. Theweak intracellular band that can be observed at 100 kDa probablyrepresents ST-endoT dimers, since the ST6GalI domain is known tooligomerize.¹

FIGS. 3A and 3B: In vivo de-N-glycosylation by transient transfection ofthe endoT fusion constructs. To evaluate the de-N-glycosylation by theendoT fusion proteins, the fusion constructs were transientlytransfected to 293 SGnTI−/− cells that stably and inducibly expressedthe Flt3 receptor extracellular domain (Flt3ECD, FIG. 3A) or to 293SGnTI−/− cells stably and inducibly expressing the 5-hydroxy-tryptaminreceptor 1D (5HT1D, FIG. 3B). Samples were analyzed by immunoblotting todetect the C-terminal HIS-tag (FIG. 3A) or the C-terminal Rho1D4 tag(FIG. 3B). The numbers in both FIGS. 3A and 3B represent samples fromcells transfected with 1=empty plasmid, 2=s-endoT plasmid, 3=GM₂S-endoTplasmid, 4=ST-endoT plasmid. Letters a and b in FIG. 3A representsample/supernatant 48 and 72 hours after transfection/induction. The +sign indicates purified Flt3ECD as a positive control. It is evidentfrom these blots that both Flt3ECD and 5HT1D samples show a reduction inmolecular weight upon transfection of any of the endoT constructs (2, 3,4), but not with the empty plasmid (1), indicating de-N-glycosylation bythe endoT fusion constructs. Clearly, endoT can deglycosylateco-expressed glycoproteins whether it is retained intracellularly(ST-endoT) or not (s-endoT and GM₂S-endoT).

FIG. 4: ConA sensitivity assay² for two ST-endoT overexpressing clonesand the parental 293 SGnTI−/− line. A lectin sensitivity assay wasperformed to determine the ConA sensitivity of 293SGnTI−/− cells and twoendoT overexpressing clones. Both clones were much more resistant toConA than the parental line (293 SGnTI−/−: 2 μg/ml). However, the firstclone was more resistant to ConA (>22 μg/ml) than the second clone (18μg/ml), and was thus selected for further work. It was designated 293SGlycoDelete. The stability of 293 SGlycoDelete line resistance to ConAwas tested over 20 splits (#+8 vs. #+28). Resistance/sensitivity wasfound to be stable and >20 μg/ml (data not shown). Higher concentrationsthan about 20 μg/ml could not be tested, because aggregates started toform.

FIGS. 5A and 5B: Validation of endoT by PCR and Western blot. FIG. 5A:PCR validation of the presence of the ST-endoT coding sequence in 293SGlycoDelete cells genomic DNA (gDNA). Analysis of the PCR products bycapillary electrophoresis illustrates the presence of a specific PCRproduct of the expected length (346 bp) with 293 SGlycoDelete gDNA asthe template (arrow). This amplicon is not generated with 293SGnTI−/−gDNA as the template for the PCR reaction. FIG. 5B: Samples from293SGnTI−/− and 293SGlycoDelete cells were analyzed by immunoblotting todetect the presence of endoT catalytic domain (polyclonal rabbitanti-endoT). The main band in the 293 SGlycoDelete cell lysate runs atthe expected MW of monomeric ST-endoT (49.8 kDa). Bands at approximately100 and 200 kDa in the 293SGlycoDelete cell lysate are probablyoligomers, while bands at lower MW likely represent degradationproducts. The oligomers are also observed in transient transfectionexperiments with the ST-endoT construct (FIG. 2). No signals for thesebands can be detected in the 293 SGnTI−/− lysate.

FIGS. 6A and 6B: Comparative expression scatterplots of the S-lineagecell lines. Values represent the mean log 2 signal intensities ofexpressed genes as determined after background correction and removal ofnoise. FIG. 6A: 293 SGnTI−/− versus 293 S. The correlation coefficientis 0.947. From the 7526 expressed genes, 68 were found to besignificantly differentially expressed (p<0.01) with at least a two-foldchange in expression in the 293SGnTI−/− line compared with 293S. FIG.6B: 293 SGlycoDelete vs 293S. The correlation coefficient is 0.938. Fromthe 7473 expressed genes, 70 were found to be significantlydifferentially expressed (p<0.01) with at least a two-fold change inexpression in the 293 SGlycoDelete line compared with 293 S. Of thesegenes, 45 (−/+65%) are the same for both the derived cell lines versusthe parental 293S cells.

FIGS. 7A-7F: GlycoDelete glycan characterization. FIG. 7A: SDS-PAGE ofGM-CSF samples from 293S, 293 SGnTI(−) and 293 SGlycoDelete cells. Eachsample was treated with PNGaseF, sialidase or both, analyzed on anSDS-PAGE gel and stained with Coomassie Brilliant Blue. kDa, kilodalton.FIG. 7B: MALDI-time-of-flight-MS spectra of GM-CSF samples (SEQ IDNO:25). Peaks are labeled with their mass/charge ratio (m/z) values. Thespectrum of the 293 SGnTI(−) GM-CSF reveals the presence of Man5GlcNAc2(left) and fucosylated Man5GlcNAc2 (right) on the glycopeptidecontaining N37. These glycoforms are absent in GlycoDelete GM-CSF. Newpeaks at m/z values corresponding to HexNAc, Hex-HexNAc andNeu5Ac-Hex-HexNAc-modified glycopeptides are detected. Spectra ofexoglycosidase-digested GlycoDelete GM-CSF N-glycans withα-2,3-sialidase or both a broad spectrum sialidase andβ-1,4-galactosidase are shown. These spectra show that N-glycans onGlycoDelete GM-CSF N37 are Neu5Ac-α-2,3-Gal-β-1,4-GlcNAc andGal-β-1,4-GlcNAc. FIG. 7C: ThermoFluor assay of GM-CSF produced by 293S,293 SGlycoDelete and E. coli cells. Similar average (n=3) melting curveswere observed for all GM-CSF glycoforms (Tm is ˜60° C.). FIG. 7D:Bioactivity of 293 S- and 293 SGlycoDelete-produced GM-CSF as measuredin a TF1 erythroleukemia cell-proliferation assay (n=3). E.coli-produced GM-CSF serves as a non-glycosylated control sample. Errorbars, s.d. (Table 5). FIG. 7E: ELISA analysis of anti-glycan antibodytiters in GlycoDelete GM-CSF immunized rabbit serum. Removal of sialicacid and galactose monosaccharides from the GlycoDelete glycan does notreduce serum antibody recognition (Table 6). FIG. 7F: duplicateexperiment as described in FIG. 7E.

FIG. 8: MALDI-TOF-MS of GM-CSF glycopeptides. Glycopeptides encompassingAsn 27 in both lines, showing the presence of Man5GlcNAc2-Asn(m/z=1931.6) and fucosylated Man5GlcNAc2-Asn (m/z=2077.7) in GnTI−/−GM-CSF (Row A). These glycoforms are absent in GlycoDelete GM-CSF (RowB). Peaks at m/z=918.5, 1080.5 and 1371.6 are detected in GlycoDeleteGM-C SF, representing HexNAc-Asn, Hex-HexNAc-Asn and Sia-Hex-HexNAc-Asn,respectively. Analysis of exoglycosidase-digested GlycoDelete GMCSFN-glycans with α-2,3-sialidase (Row C) or both a broad spectrum A.ureafaciens sialidase and S. pneumoniae β-1,4-galactosidase (Row D) areshown. The spectra illustrate that the N-glycans on GlycoDelete GM-CSFare Neu5Ac-α-2,3-Gal-β-1,4-GlcNAc-Asn and Gal-β-1,4-GlcNAc-Asn.

FIG. 9: DSA-FACE analysis of GM-CSF produced in 293S cells. Row a:dextran ladder reference. Row b: DSA-FACE profile of untreated GM-CSFproduced in 293S cells with annotated structures. Glycosylation ofGM-CSF produced in 293S cells results in a heterogeneous mix of mainlydi-, tri-, and tetra-antennary fucosylated complex type N-glycanswithout galactosylation. At lower electrophoretic mobility, somegalactosylated structures are observed. Row c: Galactosylated structuresdisappear from the spectrum upon galactosidase digestion. Row d: Twominor annotated peaks at the highest electrophoretic mobility collapseto a single peak with even higher electrophoretic mobility aftermannosidase digestion. No further major changes occur after mannosidasedigestion, indicating little terminal mannose residues are exposed. Rowe: Most annotated peaks shift to two peaks at high electrophoreticmobility upon hexosaminidase treatment. The minor peak represents thenon-fucosylated core N-glycan; the major peak represents the fucosylatedtrimannosyl core N-glycan. Row f: Core fucosylation for the majority ofthe N-glycans was observed. This is illustrated by a shift toward higherelectrophoretic mobility of many of the observed peaks after fucosidasetreatment of the glycans. Row g: No major changes were observed in theglycan profile upon treatment with a broad-spectrum sialidase,suggesting the absence of sialylation in the glycans of GM-CSF producedin 293S cells.

FIG. 10: MALDI-TOF-MS analysis of hGM-CSF produced in 293S GlycoDeleteand 293S cells. Row 1: 293 S-produced hGM-CSF. The enormous observedheterogeneity is largely due to the variability of 293S N-glycosylation.Row 2: hGM-C SF sialidase digest results in some heterogeneityreduction. Row 3: hGM-CSF digested with PNGaseF has a strongly reducedheterogeneity, demonstrating that N-glycosylation is the main source ofmolecular weight heterogeneity. Row 4: 293 SGlycoDelete produced hGM-CSFhas a strongly reduced heterogeneity. Row 5: Sialidase digest on 293SGlycoDelete produced hGM-CSF reveals a pattern of similarly lowcomplexity as the completely de-N-glycosylated 293 S-produced protein.

FIG. 11: Immunoblotting of 5HT1DR produced in 293SGnTI−/− and 293SGlycoDelete cells. Treatment of membrane protein extracts with PNGaseFrevealed a large shift in the molecular weight (MW) of the 5HT1DR stablyproduced n 293 SGnTI−/− cells (1), as expected. Contrary to this,receptor produced in 293 SGlycoDelete cells (2) did not shift in MW uponPNGaseF treatment and ran at approximately the same MW as deglycosylated(PNGaseF-treated) receptor from 293 SGnTI−/− cells. This is consistentwith a complete removal of the 5HT1DR N-glycans in the 293S GlycoDeletecells.

FIG. 12: Immunoblot analysis of anti-CD20 produced in 293S or 293SGlycoDelete cells. Equal volumes of culture medium of 293S wild-typecells and 293 SGlycoDelete cells upon transient transfection usingidentical methods were analyzed by immunoblotting. Consequently, theblot shows the level of protein expression of the anti-CD20 monoclonalantibody in the culture medium. The yield of the recombinant protein issimilar for both cell lines, indicating that the genetic manipulationsused to derive GlycoDelete 293 cells from the WT 293S precursors do notsubstantially affect the cell's capacity of protein secretion.

FIG. 13: DSA-FACE analysis of anti-CD20 produced in 293S cells. Row a:dextran ladder reference. Row b: DSA-FACE profile of untreated anti-CD20produced in 293S cells with annotated structures. Glycosylation ofanti-CD20 produced in 293S cells results in core-fucosylated diantennaryN-glycans with or without galactosylation. Row c: No major changes wereobserved in the glycan profile upon treatment with a broad-spectrumsialidase, suggesting the absence of sialylation in the glycans ofanti-CD20 produced in 293S cells. Row d: Galactosylated structuresdisappear from the spectrum upon galactosidase digestion. A single peakremains, representing the non-galactosylated core-fucosylateddiantennary N-glycan. Row e: Core fucosylation for all detectedN-glycans was observed. This is illustrated by a shift toward higherelectrophoretic mobility of the observed peaks after fucosidasetreatment of the glycans.

FIGS. 14A-14G: Functional and immunological characterization ofGlycoDelete anti-CD20. FIG. 14A: SDS-PAGE of anti-CD20 from 293SGlycoDelete (Gl.Del) and 293S cells. On the left, “PNGase” indicatesthe PNGase enzyme band. HC, antibody heavy chain; LC, antibody lightchain; kDa, kilodalton. FIG. 14B: LC-MS/MS in SRM mode of GlycoDeleteanti-CD20 glycopeptides. Peak labels state LC elution times (minutes).Trisaccharide-, disaccharide- and monosaccharide-modified glycopeptidesare shown in red, blue and yellow, respectively. Exoglycosidase digestswith sialidase and β-1,4-galactosidase illustrate identical glycans asobserved for GM-CSF. FIG. 14C: CD20-binding by anti-CD20 as assessed byflow cytometry (Table 7). FIG. 14D: Average melting curves (n=3) asdetermined in a ThermoFluor assay for untreated or PNGaseF-digested 293Sand 293 SGlycoDelete anti-CD20. FIG. 14E: Competition ELISA (top three)and ADCC assay (bottom) to assess effector function of the anti-CD20 Fc.Concentration series of 293S and 293 SGlycoDelete anti-CD20 comparingcompetition with a coated anti-Fc antibody. Error bars (ELISA), s.e.m.(n=3). Error bars (ADCC), s.d., n=3 (Table 8). FIG. 14F: Anti-glycanantibody ELISA analysis of 293 SGlycoDelete anti-CD20 immunized rabbitserum. Analysis of anti-CD20 recognition by antibodies in the serum ofrabbits immunized with GlycoDelete GM-C SF. Anti-CD20 samples weretreated with sialidase, sialidase and galactosidase or no enzyme. Errorbars, s.d., n=3 (Table 9). FIG. 14G: Blood concentrations of anti-CD20measured over time after intravenous injection of 293S or 293SGlycoDelete anti-CD20. Error bars, s.e.m., n=4. Numerical data for thisgraph are in Table 10.

FIGS. 15A-15C: LC-MS analysis of anti-CD20 hIgGl produced in 293S (left)and 293 SGlycoDelete (right) cells. FIG. 15A: deconvoluted ESI spectrumfor the reduced heavy chain, which carries the single N-glycosylationsite. For 293 S-produced anti-CD20, the typical core-fucosylatedagalacto-, mono-, and bi-galactosylated biantennary glycans are thedominant species, while a low amount of Man5Gn2 N-glycan is alsodetected. Sialylation is almost undetectable. For the 293 SGlycoDeleteanti-CD20, HexNAc-Asn, Hex-HexNAc-Asn and NeuNAc-Hex-HexNAc-Asn dominatethe spectrum, while a minor fraction of Man5Gn2 is also formed herein.Importantly, no non-N-glycosylation-related heterogeneity is detectable,supporting the notion that GlycoDelete manipulation of HEK293 cells doesnot lead to the unexpected induction of other post-translationalmodification pathways. FIG. 15B: The light chain was unaffected by theGlycoDelete engineering as it carries no N-glycosylation sites. FIG.15C: Deconvoluted mass spectra for the intact, nonreduced antibody. Allspecies can be interpreted as a combinatorial series of the glycoformson both heavy chains. In both antibodies, the number of S-S bridges iscalculated as 12-13, based on the difference in mass between the reducedchains and the assembled antibody.

FIG. 16: Size exclusion chromatography of anti-CD20. Size exclusionchromatography of 293S anti-CD20 (blue line) and 293 SGlycoDeleteanti-CD20 (red line). Only the monomeric peak is detected indicatingthat there is no aggregation in both glycoforms.

FIG. 17: Anti-CD20 pharmacokinetics in mice. Repeat experiment in anindependent laboratory from the experiment shown in FIGS. 14A-14G of themain text, also including earlier time points post-injection. Beforereaching the peak concentration in the blood, less of the anti-CD20 isremoved, resulting in increased circulating levels. The subsequent slowclearance (beyond 1 hour post-injection) is comparable for bothglycoforms, as also observed in the experiment reported in FIGS.14A-14G.

FIG. 18: GlycoDelete produced Etanercept Fc-chain glycan analysis. Datashown for one run are representative of three runs.

DETAILED DESCRIPTION Definitions

This disclosure will be described with respect to particular embodimentsand with reference to certain drawings but the disclosure is not limitedthereto but only by the claims. Any reference signs in the claims shallnot be construed as limiting the scope. The drawings described are onlyschematic and are non-limiting. In the drawings, the size of some of theelements may be exaggerated and not drawn on scale for illustrativepurposes. Where the term “comprising” is used in the present descriptionand claims, it does not exclude other elements or steps. Where anindefinite or definite article is used when referring to a singularnoun, e.g., “a,” “an,” or “the,” this includes a plural of that noununless something else is specifically stated.

Furthermore, the terms “first,” “second,” “third,” and the like, in thedescription and in the claims, are used for distinguishing betweensimilar elements and not necessarily for describing a sequential orchronological order. It is to be understood that the terms so used areinterchangeable under appropriate circumstances and that the embodimentsof the disclosure described herein are capable of operation in othersequences than described or illustrated herein.

The following terms or definitions are provided solely to aid in theunderstanding of the disclosure. Unless specifically defined herein, allterms used herein have the same meaning as they would to one skilled inthe art of this disclosure. Practitioners are particularly directed toSambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Press, Plainsview, N.Y. (1989); and Ausubel et al.,Current Protocols in Molecular Biology (Supplement 47), John Wiley &Sons, New York (1999), for definitions and terms of the art. Thedefinitions provided herein should not be construed to have a scope lessthan understood by a person of ordinary skill in the art.

A “higher eukaryotic cell” as used herein refers to eukaryotic cellsthat are not cells from unicellular organisms. In other words, a highereukaryotic cell is a cell from (or derived from, in case of cellcultures) a multicellular eukaryote. Typically, the higher eukaryoticcells will not be fungal cells. Even more typically, the highereukaryotic cells will not be plant cells or fungal cells. Particularly,the term refers to animal cells (or, typically, cell lines, such asinsect cell lines or mammalian cell lines). More particularly, the termrefers to vertebrate cells, even more particularly to mammalian cells.The higher eukaryotic cells as described herein will typically be partof a cell culture (e.g., a cell line, such as an HEK or CHO cell line),although this is not always strictly required (e.g., in case of plantcells, the plant itself can be used to produce protein).

An “endoglucosaminidase” or “endoglucosaminidase enzyme” as used hereinrefers to enzymes that hydrolyze the bond between the anomeric carbon ofa non-terminal beta-linked N-acetylglucosamine residue in anoligosaccharide of a glycoprotein or a glycolipid, and its aglycon,thereby releasing mono- or oligosaccharides from glycoproteins orglycolipids or sugar polymers. Endoglucosaminidases are a subset of theglycosidases, and may or may not have other enzymatic activities (suchas, e.g., glycosyltransferase activity). A particular class ofendoglucosaminidases is formed by the endo-β-N-acetylglucosaminidases ormannosyl-glycoprotein endo-P-N-acetylglucosaminidases, indicated as EC3.2.1.96 in the International Union of Biochemistry and MolecularBiology (IUBMB) nomenclature. This particular class of enzymes arecapable of catalyzing the endohydrolysis of the N,N′-diacetylchitobiosylunit in high-mannose glycopeptides and glycoproteins containing the-[Man(GlcNAc)2]Asn- structure. One N-acetyl-D-glucosamine (GlcNAc)residue remains attached to the protein; the rest of the oligosaccharideis released intact. Thus, the result is a single GlcNAc-modifiedglycoprotein. Of note, the remaining GlcNAc residue may be eitherunmodified or still be modified with other sugar residues in positionsother than that of the hydrolyzed bond, for instance, the GlcNAc residuemay carry a fucose on position 3 or 6. Nevertheless, glycoproteins witha modified GlcNAc residue will still be referred to as singleGlcNAc-modified proteins, as there is no second sugar residue onposition 4 of the GlcNAc residue (i.e., there is no typical sugarchain). A particular advantage of endoglucosaminidases as compared toexoglycosidases is that they allow discrimination between N-linked andO-linked glycans and between classes of glycans. A non-limiting list ofendoglucosaminidases is provided in the application.

An “Fc-containing molecule” as used in the application refers toproteins or fusion proteins that contain an Fc region. An Fc region(fragment-crystallizable region) is the tail region of an immunoglobulinthat interacts with cell surface receptors called Fc receptors and someproteins of the complement system. According to particularly envisagedembodiments, the Fc region in the Fc-containing molecule is an Fc regionfrom an immunoglobulin G (IgG) isotype. This can be any of the IgGsubclasses (IgG1, 2, 3, 4 in humans). For IgG, like IgA and IgDisotypes, the Fc region is composed of two identical protein fragments,derived from the second and third constant domains of the antibody's twoheavy chains. The Fc regions of IgGs bear a highly conservedN-glycosylation site, indicated as N297 (Asn-297 or Asparagine 297).“Fc-containing molecules” as used herein encompass both proteins thatnaturally have an Fc region (such as immunoglobulins), or fusionproteins or molecules, wherein an Fc region is fused to a protein,peptide or other molecule (particularly a binding moiety). Examples ofFc-fusion proteins include, but are not limited to, those described inHuang, 2009. Of note, an Fc molecule as such is also an Fc-containingmolecule. A particular class of Fc-containing molecules areFc-containing molecules that can bind antigen. Examples are antibodies,or fusion proteins wherein an Fc region is linked to a binding moiety(e.g., a nanobody, a Fab region, a F(ab')2 region).

Typically, the Fc part in the Fc-containing molecules will be a human orhumanized sequence, meaning that the amino acid sequence of the Fcregion is at least 95% identical to a human Fc sequence, particularly atleast 99% identical to a human Fc sequence, or most particularly is 100%identical to a human Fc sequence. However, the disclosure is not limitedto human sequences. For instance, it is possible that the Fc region isthat of a mouse, or of a camelid, a rhesus monkey, a dog, a cow, aguinea pig, a sheep, a goat, a horse, a rat, a rabbit, a cat, or anyother mammal. It is even possible that the Fc region is fromnon-mammalian animals (e.g., a chicken). In such cases, the skilledperson will understand that, while the N-glycosylation site is conservedacross species, the exact position may differ and is not always N297.Using a simple sequence alignment, the right residue can be identified,if necessary.

A “Golgi localization signal” is a molecule, typically a peptide, thatdirects localization of the polypeptide or protein to which it isconjugated to the Golgi apparatus. Localization thus also impliesretention in the Golgi apparatus. Typically, these localization (orretention) sequences are peptide sequences derived from (pre)proteinsthat are situated in the Golgi when functionally active as a matureprotein.

The glycans and monosaccharides mentioned herein are sometimes indicatedwith their recognized abbreviations: e.g., Glc for β-D-Glucose, Man forβ-D-Mannose, Gal for β-D-Galactose, GlcNAc for β-D-N-Acetylglucosamine,GalNAc for β-D-N-Acetylgalactosamine, NeuNAc for α-N-Acetylneuraminicacid, also known as sialic acid (Sia), Fuc for α-L-Fucose, Hex forhexose.

This disclosure aims to provide higher eukaryotic cells producingFc-containing molecules with an altered glycosylation pattern, inparticular, a more homogeneous glycosylation pattern, that makes themmore amenable for further use, e.g., therapeutic use, or easierbiomanufacturing.

This is achieved, according to a first aspect, by providing highereukaryotic cells, particularly animal cells, with a first exogenousnucleic acid sequence encoding an endoglucosaminidase enzyme and asecond exogenous nucleic acid sequence encoding an Fc-containingmolecule.

According to particular embodiments, the higher eukaryotic cell isglyco-engineered to be deficient in synthesizing complex type sugars(and may or may not be engineered to be deficient in synthesizing hybridtype glycans). More particularly, the higher eukaryotic cell is a highereukaryotic cell only capable of producing high mannose N-glycans. Thiscan be achieved, e.g., by making the cell deficient inN-acetylglucosaminyltransferase 1 activity. According to particularembodiments, the glycosyltransferase GnTI, encoded by the gene MGAT1(Gene ID: 4245 in humans), is inactivated in the cells.

Accordingly, higher eukaryotic cells incapable of synthesizing complextype or hybrid type N-glycans are provided, additionally characterizedby having a first exogenous nucleic acid sequence encoding anendoglucosaminidase enzyme and a second exogenous nucleic acid sequenceencoding an Fc-containing molecule. For example, higher eukaryotic cellsdeficient in N-acetylglucosaminyltransferase 1 activity are provided,additionally characterized by having a first exogenous nucleic acidsequence encoding an endoglucosaminidase enzyme and a second exogenousnucleic acid sequence encoding an Fc-containing molecule.

Higher eukaryotic cells can be of any higher eukaryotic organism, but inparticular embodiments, mammalian cells are envisaged. The nature of thecells used will typically depend on the desired glycosylation propertiesand/or the ease and cost of producing the glycoprotein. Mammalian cellsmay, for instance, be used to avoid problems with immunogenicity. Highereukaryotic cell lines for protein production are well known in the art,including cell lines with modified glycosylation pathways. Non-limitingexamples of animal or mammalian host cells suitable for harboring,expressing, and producing proteins for subsequent isolation and/orpurification include Chinese hamster ovary cells (CHO), such as CHO-K1(ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec. Genet.,12:555-556; and Kolkekar et al., 1997, Biochemistry 36:10901-10909),CHO-K1 Tet-On cell line (Clontech), CHO designated ECACC 85050302 (CAMR,Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B(GEIMG, Genova, IT), CHO-K1/SF designated ECACC 93061607 (CAMR,Salisbury, Wiltshire, UK), RR-CHOK1 designated ECACC 92052129 (CAMR,Salisbury, Wiltshire, UK), dihydrofolate reductase negative CHO cells(CHO/-DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. U.S.A.77:4216), and dp12.CHO cells (U.S. Pat. No. 5,721,121); monkey kidneyCV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651); humanembryonic kidney cells (e.g., 293 cells, or 293T cells, or 293 cellssubcloned for growth in suspension culture, Graham et al., 1977, Gen.Virol. 36:59); baby hamster kidney cells (BHK, ATCC CCL-10); monkeykidney cells (CV1, ATCC CCL-70); African green monkey kidney cells(VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse sertoli cells (TM4,Mather, 1980, Biol. Reprod. 23:243-251); human cervical carcinoma cells(HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lungcells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mousemammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat liver cells(BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals N.Y. Acad. Sci.383:44-68); MCR 5 cells; FS4 cells. According to particular embodiments,the cells are mammalian cells selected from CHO cells, Hek293 cells orCOS cells. According to further particular embodiments, the mammaliancells are selected from CHO cells and Hek293 cells.

It is particularly envisaged that the endoglucosaminidase enzymeproduced by the higher eukaryotic cell will act on the Fc-containingmolecule produced in the cell, and removes the N-glycosylation.According to particular embodiments, the endoglucosaminidase enzymeencoded by the first exogenous nucleic acid sequence is amannosyl-glycoprotein endo-beta-N-acetylglucosaminidase, i.e., it hasthe activity of E.C. 3.2.1.96 in the IUBMB nomenclature, implying thatit can remove sugar chains while leaving one GlcNAc residue on theprotein (importantly, it also acts on the common core pentasaccharideMan₃GlcNAc₂). According to alternative embodiments, theendoglucosaminidase encoded by the first exogenous nucleic acid sequencehas different affinities toward different types of glycosylationstructures. Typical examples of the latter are endoglucosaminidases thatare able to hydrolyze hybrid type sugars and/or high-mannose sugars, butare not capable of cleaving complex type glycans. According to furtherparticular embodiments, the endoglucosaminidase is amannosyl-glycoprotein endo-beta-N-acetylglucosaminidase that hasdifferent affinities toward different types of glycosylation structures.According to yet further particular embodiments, theendo-beta-N-acetylglucosaminidase is able to cleave hybrid-type sugarsand/or high-mannose sugars, but not complex-type glycans. According toeven more particular embodiments, the endoglucosaminidase is EndoH orEndoT. According to most particular embodiments, the endoglucosaminidaseis Endo T.

To ensure that the endoglucosaminidase effectively removes the sugarchains of the Fc-containing protein, it is envisaged that theendoglucosaminidase not only remains in the cell, but is also fullyactive. Its activity should be regulated spatiotemporally in order toensure that the desired hydrolysis takes place. Thus, according toparticular embodiments, the expression of the endoglucosaminidase enzymeis targeted to the Golgi apparatus. This can be achieved by operablylinking the endoglucosaminidase to a Golgi localization signal. Suchsignal directs the endoglucosaminidase to the Golgi, where it isretained. As the Golgi apparatus is, next to the ER, the intracellularlocation where glycosylation of proteins takes place, targeting to thisorganelle ensures that the endoglucosaminidase is in the correctintracellular position to modify the glycosylation of the glycoprotein.

This is particularly beneficial for controlling the furtherglycosylation, as the higher eukaryotic cells possess further enzymesneeded for complex glycosylation that are also present in the Golgisecretory pathway. Indeed, the endoglucosaminidase can be targeted insuch a way that these enzymes act cooperatively on the Fc-containingmolecule. In higher eukaryotic cells, the luminal surface of the ER andGolgi apparatus provides catalytic surfaces that allow the sequentialprocessing of glycoproteins as they proceed from the ER through theGolgi network into the medium. As a glycoprotein (such as theFc-containing molecule) proceeds from the ER through the secretorypathway, it is sequentially exposed to different mannosidases andglycosyltransferases. Several processing steps rely on previousreactions because some N-glycosylation enzymes depend on a particularsubstrate that is created by the previous enzyme. N-glycosylationenzymes, in particular, exogenous enzymes such as theendoglucosaminidase, must, therefore, be arranged in a predeterminedsequence to allow for the synthesis of specific N-glycan structures.

However, while the cells described herein are particularly useful toproduce the desired Fc-containing molecules with the right glycosylationpattern, one should keep in mind that it is also possible to produce andadd all or part of the desired sugar profile synthetically, in vitro(e.g., by enzymatic coupling on the produced (optionally deglycosylated)protein).

Establishing the sequential processing environments of the secretorypathway requires the proper localization of N-glycosylation enzymes. Themechanisms by which secreted proteins can be transported through thesecretory pathway (from the ER to the cis-, medial- and trans-Golgicompartments and into the medium), while each compartment maintains aspecific set of resident (for example, N-glycosylation) enzymes, hasbeen the subject of extensive study. Two well-established mechanismsthat localize proteins to the various compartments of the secretorypathway are retrieval and retention (van Vliet et al., PBMB 1 2003;Teasdale et al., 27 1996).

Retrieval is a process by which proteins are localized to certainorganelles through interaction with other proteins. Several ER-residingproteins contain a carboxy-terminal tetrapeptide with the consensussequence KDEL (SEQ ID NO:23) (or HDEL (SEQ ID NO:24) in yeast), whichhas been shown to be required for efficient localization to the ER.

Several ER- and Golgi-residing enzymes are type II membrane proteins.These proteins have a common domain structure comprising a shortcytoplasmic tail at the amino terminus, a hydrophobic transmembranedomain, a luminal stem and a C-terminal catalytic domain. Deletionstudies as well as fusions to non-Golgi-residing proteins haveidentified the N-terminus, and, in particular, the transmembrane region,as containing the targeting information of many type II membraneproteins. Although it is clear that N-terminal domains are involved intargeting, the extent to which their targeting ability is transferablebetween different species is not yet totally clear. Nevertheless,considerable advances have been made, such as the design of geneticlibraries of known type II membrane protein domains that encode peptidesthat are associated with proteins that naturally localize to the ER andGolgi of S. cerevisiae or P. pastoris (Choi et al., 5022 (2003);Hamilton et al., Science 1244) confirming the suitability of, e.g., theleader sequence from S. cerevisiae Sec12 (ER localization), MNN2 (Golgilocalization), and MNN9 (Golgi localization). Sequences listed in Table5 of WO02/000879 include HDEL and the leader sequences from Mnsl for ERlocalization, and leader sequences from Och1 and Mnt1 (Golgi-cislocalization), from Mnn2 (Golgi medial localization), from Mnn1 (Golgitrans localization), from alpha-2,6-sialyltransferase (trans-Golginetwork) and from beta-1,4-galactosyltransferase I (Golgi localization).

Localization signals thus are well known in the art and may be derivedfrom proteins that are normally localized in the ER or Golgi for theirfunction. Moreover, localization sequences from one organism mayfunction in other organisms. For example, the membrane spanning regionof α-2, 6-sialyltransferase from rats, an enzyme known to localize inthe rat trans Golgi, was shown to also localize a reporter gene(invertase) in the yeast Golgi (Schwientek, et al., 1995). Schwientekand co-workers have also shown that fusing 28 amino acids of a yeastmannosyltransferase (Mnt1), a region containing an N-terminalcytoplasmic tail, a transmembrane region and eight amino acids of thestem region, to the catalytic domain of human GalT are sufficient forGolgi localization of an active GalT (Schwientek et al. (1995), J. Biol.Chem. 270 (10): 5483-5489). Other well-documented motifs are the KDELand HDEL motif for retention in the ER. According to particularembodiments, the ER or Golgi localization signal is from a protein thatis itself localized in the ER or Golgi when functionally active.Examples of such proteins include, but are not limited to, S. cerevisiaedipeptidyl aminopeptidase A (Ste13p), human β-galactoside-α-2,6-sialyltransferase (ST6GalI) and the human gangliosi de-GM2-synthase.According to further embodiments, the localization sequence is derivedfrom one of the following proteins: Ste13p, GL2-synthase,ganglioside-GM2-synthase, and α-2,6-glycosyltransferase, in particular,α-2,6-sialyltransferase, most particularlyβ-galactoside-α-2,6-sialyltransferase.

Importantly, the Golgi apparatus is not just one homogeneous region, buthas five functional regions: the cis-Golgi network, cis-Golgi,medial-Golgi, trans-Golgi, and trans-Golgi network. Vesicles from theendoplasmic reticulum (via the vesicular-tubular cluster) fuse with thecis-Golgi network and subsequently progress through the stack ofcisternae that make up the Golgi apparatus to the trans-Golgi network,where they are packaged and sent to the required destination. Eachregion contains different enzymes that selectively modify the contents,e.g., depending on where they are destined to reside. Thus, depending onthe exact targeting of the endoglucosaminidase within cells,glycosylation pathways may be modified in different ways.

While the endoglucosaminidase may be targeted late in the Golgi, toprovide an “in vivo clean-up” of aberrantly glycosylated proteins, aparticularly envisaged modification is targeting the endoglucosaminidaseto an earlier stage in the Golgi glycosylation pathway, while one ormore glycosyltransferases (typically endogenous glycosyltransferases inthe case of higher eukaryotic cells, although exogenousglycosyltransferases are envisaged as well) are active furtherdownstream. This way, a uniform glycopopulation (e.g., of singleGlcNAc-modified Fc-containing molecules) is presented as substrate tothe glycosyltransferases. This results in a uniform population ofglycosylated Fc-containing molecules. Note that this uniformglycopopulation may particularly be a uniform population ofnon-naturally occurring glycoforms, as typical endoglucosaminidases willalso remove the inner Man3GlcNAc2 core structure typical of naturalglycostructures. However, such structures are often less immunogenic inmammals than particular glycans produced in plant, yeast or insectcells. As shown in the Examples section, a particularly envisagedtargeting is targeting in the Golgi so that endogenousgalactosyltransferase and sialyltransferase act sequentially on theprotein, e.g., by targeting the endoglucosaminidase to the trans-Golgi.The sequential action of these enzymes yields trisaccharide structureson the produced Fc-containing molecules: a GlcNAc closest to theglycosylated asparagine residue, coupled to a Gal moiety and ending in aNeuNAc (sialic acid) moiety.

The Fc-containing molecules produced by the cells described hereintypically should be easily recovered. This will particularly be achievedby secretion of the Fc-containing molecules. This may happenspontaneously, or by addition of a secretion signal. The nature of thesecretion signal will typically not depend on the protein to besecreted, but on the type of higher eukaryotic cells used. As long asthe secretion signal is functional in the cell type in which it is used(i.e., it results in secretion to the extracellular environment of theprotein or peptide to which it is fused), this feature is not criticalto the disclosure. Thus, secretion signals from other organisms may beused, as long as these signals lead to secretion in the highereukaryotic cells used. Secretion signals are well known in the art andmay be derived from—typically the N-terminus of—proteins that aresecreted, or may be made synthetically (e.g., Tan et al., ProteinEngineering 2002, vol. 15, no. 4, pp. 337-345). Alternatively, they canbe derived from genomic sequences using computational methods (Klee etal., BMC Bioinformatics 2005, 6:256). Also, bacterial secretion signalscan be used. Further examples of signal peptides that can be used aredescribed in WO2002/048187 (eukaryotic cells), Schaaf et al. (BMCBiotechnol. 2005; 5: 30) (moss cells), EP549062.

The glycosylation status of the produced Fc-containing molecule willdepend both on the cellular system used (e.g., which enzymes are presenttherein) and the specificity of the endoglucosaminidase. Moreover, thetime and place where these enzymes act is also important (e.g., whichenzyme acts first in the ER→Golgi pathway). The Fc-containing moleculesproduced in these cells can be further modified after production, e.g.,by treatment with glycosyltransferases, resulting in proteins with thedesired glycan moieties. However, it is particularly envisaged to usecells capable of producing Fc-containing molecules with specific glycanmoieties, namely those with a GlcNAc-Gal-NeuNAc trisaccharide structure(with GlcNAc bound to the Asparagine residue of the Fc-containingmolecule, in particular, the N297 residue of IgG Fc-containingmolecules). Typically, this is achieved by eliminating the capacity forsynthesizing complex sugars on the exogenous Fc-containing molecule(e.g., by eliminating N-acetylglucosaminyltransferase 1 activity), andtargeting the exogenous endoglucosaminidase to the Golgi network, whereit acts before the galactosyltransferase and the sialyltransferase. Thiseliminates the need for further glycosyltransferase treatment afterproduction, as Fc-containing molecules with this specific sugar profilehave beneficial properties. It is shown herein that molecules with thisspecific sugar structure are non-immunogenic, retain antigen binding,and have long-circulating half-life in vivo, while the simpleglycosylation path results in a pool of proteins with much reducedheterogeneity in glycan profiles.

Thus, the higher eukaryotic cells described herein are particularly wellsuited for production of Fc-containing molecules. It is envisaged thatFc-containing molecules produced in these cells fall within the presentscope.

Thus, according to particular embodiments, Fc-containing molecules areprovided that are obtainable by producing them in higher eukaryoticcells, wherein the cells have:

-   -   A first exogenous nucleic acid sequence encoding an        endoglucosaminidase enzyme;    -   A second exogenous nucleic acid sequence encoding the        Fc-containing molecule.

According to further particular embodiments, the endoglucosaminidaseenzyme is targeted to the Golgi apparatus (e.g., by operably linking itto a Golgi localization signal). According to alternative, non-exclusiveembodiments, the higher eukaryotic cells are glyco-engineered to beincapable of complex-type glycosylation, while retaining expression ofgalactosyltransferase and sialyltransferase. According to specificembodiments, the glyco-engineering to be incapable of complex typeglycosylation entails the inhibition or knock-down ofN-acetylglucosaminyltransferase 1.

The Fc-containing molecules obtainable by producing them in these cellsare Fc-containing molecules with a more homogeneous glycan profilecompared to those produced in higher eukaryotic cells without theendoglucosaminidase (and with the capacity for complex glycosylation).However, most often, not all molecules will have the exact sametrisaccharide sugar chain, as Fc-containing molecules that areincompletely glycosylated will also be produced. These forms carryeither a single GlcNAc moiety, or a disaccharide Gal-GlcNAc (with GlcNAclinked to an asparagine of the Fc region). However, such population ofidentical Fc-containing molecules with trisaccharide, disaccharide ormonosaccharaide structure described herein, also shows the beneficialeffects. Thus, a plurality of Fc-containing molecules obtainable byproducing them in these cells is also envisaged.

The beneficial properties of these molecules are not limited to thosemolecules produced in the cells described herein. As shown in theExamples section, the properties (in particular, the longer half-life incirculation) arise purely out of the specific glycosylation pattern. Inother words, Fc-containing molecules that have the same glycosylationstructure that is partly or wholly synthesized on the Fc-containingmolecule in vitro (e.g., by treatment of endoglucosaminidase and/orglycosyltransferase(s)) will have the same properties as those producedcompletely in the cells described herein.

Thus, Fc-containing molecules are provided, characterized in that theglycosylation on asparagines in the Fc part consists of a glycanselected from the following: a trisaccharide structureNeu5Ac-Hex-HexNAc, a disaccharide structure Hex-HexNAc, and amonosaccharide structure HexNAc (each with HexNAc linked to theasparagine). More particularly, the glycosylation will be selected fromthe trisaccharide structure and the disaccharide structure. Mostparticularly, the glycosylation will be a trisaccharide structureNeu5Ac-Hex-HexNAc.

As a pool of these Fc-containing molecules with the three differentglycosylation patterns also shows beneficial properties in vitro, aplurality of identical Fc-containing molecules are provided,characterized in that the glycosylation on asparagines in the Fc partconsists of a glycan selected from the following: a trisaccharidestructure Neu5Ac-Hex-HexNAc, a disaccharide structure Hex-HexNAc, and amonosaccharide structure HexNAc (each with HexNAc linked to theasparagine). Particularly, at least part of the plurality ofFc-containing molecules will have a glycosylation pattern selected fromthe trisaccharide structure and the disaccharide structure. Mostparticularly, at least part of the plurality of Fc-containing moleculeswill have a glycosylation that is a trisaccharide structureNeu5Ac-Hex-HexNAc.

Particularly envisaged HexNAc moieties are GlcNAc moieties. Particularlyenvisaged Hex moieties are Gal moieties. Thus, the Hex-HexNAc moiety inthe above di- and trisaccharides particularly is a Gal-GlcNAc moiety.Most particularly envisaged is the trisaccharideNeu5Ac-α-2,3-Gal-β-1,4-GlcNAc, and the corresponding disaccharideGal-β-1,4-GlcNAc.

Particularly envisaged Fc-containing molecules are molecules thatcontain an Fc from an immunoglobulin G (IgG). IgG Fc-containingmolecules all have one conserved asparagine glycosylation site,indicated as N297 in human IgGs. Thus, IgG Fc-containing moleculesdescribed herein are characterized by the specific glycosylation patternon that N297 residue.

Most (therapeutic) antibodies have no glycosylation sites in the Fabregion. Likewise, most Fc-fusion proteins also have no furtherglycosylation sites. It is particularly envisaged that the glycosylationof the Fc region is the only glycosylation present in the Fc-containingmolecule. Most particularly, it is envisaged that the glycosylation onN297 of IgG Fc-containing molecules is the only glycosylation present onthe Fc-containing molecule. This will ensure that the modification ofthe glycosylation (of the Fc part) does not interfere with theinteractions of the non-Fc part (e.g., the antigen binding of the Fabregion).

According to particular embodiments, the Fc-containing molecule is anantibody or an Fc-fusion protein that binds an antigen. According tofurther particular embodiments, the Fc-containing molecule is anantibody, most particularly an IgG. It may be any one of an IgG1, 2, 3,or 4; but IgG1 and IgG2 antibodies are most prevalent.

When discussing the specific glycosylation of the present Fc-containingmolecules, it is important to realize that these three sugar moleculesare the only sugar molecules present on the Fc-containing molecule. Inother words, these Fc-containing molecules do not have a coreMan3GlcNAc2 moiety. This is an important difference with the prior art.Indeed, stability of Gal-Sial structures have also been studied, butonly when attached to the core Man3GlcNAc2 moiety, and as a bifurcatedglycan (i.e., with 2 Gal-Sial antennae present). Moreover, thesestructures fixed to the core Man3GlcNAc2 have not been reported toprolong half-life; on the contrary, they are more sensitive to proteases(Raju et al., Biotechnol. Prog. 2007; 23(4):964-71)). This furtherhighlights the surprising effect observed for the present,non-bifurcated trisaccharide structure.

Given that Fc-containing molecules are most often used as therapeutics,and that the Fc-containing molecules with the specific glycosylationpresented herein have a longer half-life, without having altered antigenspecificity (i.e., for those Fc-containing molecules that bind anantigen, such as all antibodies, and most Fc-fusion proteins), thepresent molecules are well suited for use in medicine.

Accordingly, Fc-containing molecules obtainable by producing them inhigher eukaryotic cells as described herein are provided for use as amedicament. Also, Fc-containing molecules characterized by having aglycosylation on asparagines in the Fc part consists of a glycanselected from the following: a trisaccharide structureNeu5Ac-Hex-HexNAc, a disaccharide structure Hex-HexNAc, and amonosaccharide structure HexNAc (each with HexNAc linked to theasparagine), as described herein, are provided for use as a medicament.

These molecules can be used for any disorder wherein you normally woulduse Fc-containing molecules, particularly Fc-containing molecules thatbind an antigen. Since they have the same binding affinity for theantigen as their non-glycosylation modified counterparts, they have thesame applicability. Of note, as binding to Fcγ receptors is reduced bythe specific glycosylation pattern, they may be less suitable fortreating those diseases where binding of Fcγ receptors is important(e.g., Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) is thoughtto be mediated by Fcγ receptors, so the present molecules are likelyless suited to elicit this response). On the other hand, they may bemore suitable to treat those diseases where Fcγ receptor binding is notimportant or even not desired. Indeed, for antibodies that targetcell-surface molecules, especially those on immune cells, abrogatingeffector functions is required. Abrogating Fcy receptor binding proveduseful in, e.g., treatment of fetomaternal alloimmunization to the humanplatelet alloantigen-1a (Armour et al., Eur. J. Immunol. 1999;29(8):2613-24; Ghevaert et al., J. Clin. Invest. 2008; 118(8):2929-38),in treatment of autoimmune diseases or transplant rejection (Reddy etal., J. Immunol. 2000; 164(4):1925-33), in making a long-actingerythropoietin Fc-fusion protein (Yang et al., Arch. Pharm. Res. 2012;35(5):757-9), and it is envisaged that the present molecules areparticularly well suited for treating those disorders; i.e., methods oftreating these diseases in subjects in need thereof are provided,comprising administering an Fc-containing molecule as described hereinto the subj ects.

The Fc-containing molecules are also particularly suited for thosedisorders wherein a longer circulating half-life of Fc-containingmolecules is desirable, i.e., any disorder in which repeatedadministration of Fc-containing molecules is used as a therapy. Oneparticular example of such therapy is IVIG: intravenous immunoglobulin,a plasma protein replacement therapy (IgG) for immune-deficient patientswho have decreased or abolished antibody production capabilities. It isused in immune deficiencies, acquired compromised immunity conditions,autoimmune diseases, inflammatory diseases and acute infections.

Thus, the Fc-containing molecules described herein (particularly IgGmolecules as described herein) are provided for use in intravenousimmunoglobulin therapy. This is equivalent as saying that methods fortreating subjects in need of intravenous immunoglobulin therapy areprovided, comprising administering an Fc-containing molecule (IgGmolecule) as described herein to the subjects.

Of note, a standard way of prolonging half-life of Fc-containingmolecules is by increasing the affinity of the Fc-containing moleculefor the FcRn receptor (e.g., the Xtend technology by XENCOR®). As thepresent way of prolonging half-life of Fc-containing molecules isindependent of FcRn binding, the technologies are likely compatible toeven further enhance half-life.

The eukaryotic cells described herein are particularly well suited forglycoprotein production. According to particular embodiments, theglycoproteins are enriched for a specific glycoform, particularlytrisaccharide Neu5Ac-Hex-HexNAc-modified glycoproteins. Thus, methodsare provided for producing Fc-containing molecules with a specificglycosylation pattern on asparagine residues in the Fc-containingmolecule in a higher eukaryotic cell, comprising the steps of:

-   -   providing a higher eukaryotic cell comprising a first exogenous        nucleic acid sequence encoding an endoglucosaminidase enzyme,        wherein the endoglucosaminidase is operably linked to a Golgi        localization signal, and a second exogenous nucleic acid        sequence encoding the Fc-containing molecule, in conditions        suitable for expressing the endoglucosaminidase enzyme and the        Fc-containing molecule; and    -   recovering the Fc-containing molecule after it has been        intracellularly contacted with the endoglucosaminidase.

The same considerations for the cells and Fc-containing molecules applyas described above. According to a particular aspect, the proteinmodified with the single GlcNAc residue, obtained after the contactingwith the endoglucosaminidase, is only an intermediary product. Methodsaccording to this aspect will include at least one additionaltransglycosylation step. Although this transglycosylation can be doneextracellularly (via an added enzyme or via an enzyme also produced bythe cells), it is particularly envisaged that transglycosylation occursintracellularly, by glycosyltransferases expressed by the highereukaryotic cells. According to these embodiments, before the finalrecovery of the glycoprotein, the methods further involve a step ofcontacting the enzyme with one or more glycosyltransferases after it hasbeen intracellularly contacted with the endoglucosaminidase. It will beunderstood by the skilled person that, when both the endoglucosaminidaseenzyme and the one or more glycosyltransferase enzyme(s) are targeted tothe (ER or) Golgi, it is ensured that the glycosyltransferase activityoccurs after the endoglucosaminidase activity. Typically, this may beensured by targeting both enzymes to different compartments of the ER orGolgi, as there is a fixed order in which proteins follow the ER−>Golgiroute. In the event both enzymes are targeted to the same compartment,or that both activities are performed by the same enzyme, it typicallywill be ensured that the protein after the transglycosylation step is nolonger recognized as substrate for the endoglucosaminidase enzyme. Thus,separation of the enzymatic activities in time may also involve spatialseparation and/or a different substrate specificity and/or inactivationof the enzyme.

The glycosyltransferase may be encoded by an exogenous sequence, or maybe an enzyme that is endogenous in the cells having a first exogenousnucleic acid sequence encoding an endoglucosaminidase enzyme and asecond exogenous nucleic acid sequence encoding an Fc-containingmolecule.

It is particularly envisaged that the Fc-containing molecule is secretedto allow easy recovery.

A particular class of Fc-containing molecules described herein areFc-containing molecules that bind to an antigen (typically antibodies,or Fc-fusion proteins that wherein the Fc region is fused to a bindingmoiety). These molecules retain antigen binding activity and haveincreased circulation time in vivo compared to non-modified glycoforms.

Accordingly, in a further aspect, methods are provided for increasingcirculation time of an Fc-containing molecule that binds to an antigen,to be administered to a subject in need thereof, without alteringantigen binding, comprising:

-   -   providing an Fc-containing molecule with a specific        trisaccharide Neu5Ac-Hex-HexNAc-modified glycosylation pattern;    -   administering the Fc-containing molecule to the subject.

It is to be understood that although particular embodiments, specificconfigurations as well as materials and/or molecules, have beendiscussed herein for cells and methods according to this disclosure,various changes or modifications in form and detail may be made withoutdeparting from the scope and spirit of this disclosure. The followingexamples are provided to better illustrate particular embodiments, andthey should not be considered limiting the application. The applicationis limited only by the claims.

EXAMPLES Example 1 Generation of a Stable Cell Line with AlteredGlycosylation

Glycoproteins produced in mammalian cells are often heterogeneous as aconsequence of the many biosynthetic steps of complex-type N-glycansynthesis (FIG. 1A). Each step is less than 100% efficient and someenzymes compete for substrates, resulting in many different glycoforms.Therapeutic glycoprotein heterogeneity negatively impacts downstreamprocessing and process reproducibility and can lead to variable efficacysince glycans affect clearance and biological activities.^(1,2) Forinstance, the sialic acid content of the glycans often determinespharmacokinetics.³ In tackling the glycan heterogeneity problem, it hasto be considered that N-glycans are often critical for protein foldingand cannot be simply removed through N-glycosylation site mutagenesis.Here, we introduce a mammalian cell glycoengineering technology thatshortcuts the Golgi N-glycosylation pathway to produce proteins withminimal-sized sialylated trisaccharide N-glycans (FIG. 1A).

293SGnTI−/− cells⁴ produce glycoproteins modified with Man₅GlcNAc₂N-glycans. Several endo-β-N-acetylglucosaminidases⁵ are known thathydrolyze such glycans, upon which a single asparagine-linkedN-acetylglucosamine (GlcNAc) residue is left. EndoT⁶ was chosen as aeukaryotic-origin representative of this Glycoside Hydrolase family 18for expression in the mammalian cell secretory system as it has theadvantage that the pH optimum of endoT is 6.0. This is close to the pHin the mammalian trans-Golgi apparatus,⁷ but sufficiently different fromthe pH in the ER (pH 7.2), so as not to interfere substantially with theER-function of N-glycans in protein folding and quality control.Earlier, it was shown that transient Golgi-targeted expression of endoTin 293SGnTI−/− cells results in in vivo de-N-glycosylation ofglycoproteins (e.g., Examples 6 and 7 of EP2331701).

EndoT hydrolysis in the Golgi would produce single GlcNAc N-glycan“stumps” on the glycoproteins, post folding. It was speculated that suchGolgi-generated single GlcNAc residues would be recognized by the cell'sgalactosyltransferases and sialyltransferases, prior to secretion. Thiswould then result in the synthesis of the most simple sialylated type IItermini, a common element in N- and O-glycans. This three-step pathwayis much shorter than the many-step native N-glycosylation pathway andshould result in strongly reduced heterogeneity and easier N-glycancharacterization. The glycoengineering strategy described above,“GlycoDelete,” is illustrated in FIG. 1A.

In order to target endoT to the trans Golgi of 293SGnTI−/− cells, theendoT-encoding sequence was fused without its predicted signal sequenceto Golgi targeting domains from two human enzymes normally present inthe Golgi (FIG. 2). When the endoT catalytic domain was fused to thetargeting domain of the human β-galactoside-α-2,6-sialyltransferase 1(ST6GAL1)⁸ (referred to herein as the ST-endoT fusion protein), it wasretained intact in the cells. Transient expression of ST-endoT in 293SGnTI(−) cells resulted in in vivo deglycosylation of a stably expressedand secreted Flt3 receptor extracellular domain⁹ and the human5-hydroxytryptamin 1D (5HT1D) receptor (FIGS. 3A and 3B).

To establish a 293SGnTI(−)-derived cell line stably expressing ST-endoTfusion protein, cells were selected with the desired glycan phenotypeusing concanavalin A (ConA). ConA is a tetrameric cytotoxic lectin thatbinds to oligomannose and hybrid-type N-glycans. Full deglycosylation ofcell surface glycoproteins by endoT would result in the absence of ConAligands, thus rendering the cells resistant to this lectin (FIG. 1B).Four weeks after transfection, clones resistant to ConA (at the lowestconcentration that killed all of the parental 293SGnTI(−) cells) wereobtained. Two clones were selected for robust growth and subjected to aConA lectin sensitivity assay,¹⁰ and that with the highest ConAresistance was named 293SGlycoDelete (FIG. 4). Genomic integration andexpression of ST-endoT were validated by PCR and immunoblotting,respectively (FIGS. 5A and 5B). 293SGlycoDelete and 293SGnTI(−) cellshave similar morphologies, and their growth rates are indistinguishable(FIG. 1C). However, it was noticed that 293SGlycoDelete cells are lessadherent than 293SGnTI(−) cells; this is a desirable feature forsuspension cultivation, as used in biopharmaceutical production.

The transcriptomes of 293SGnTI(−) and 293SGlycoDelete cells wereprofiled using exon microarrays and it was found that only three of the7,344 genes that had detectable expression were more than two-folddifferentially expressed (P<0.01) between the two cell lines (FIG. 1D).Comparison of the 293SGnTI(−) line and the 293S parent showeddifferential transcription of about 70 genes (FIGS. 6A and 6B), withoutclear enrichment for particular pathways. Substantial genomicrearrangement was observed in the 293SGnTI(—) line (unpublishedobservations), which may account for these differences. Therefore,GlycoDelete engineering does not substantially alter the transcriptionalprofile of the cells. The absence of a transcriptional signature of theunfolded protein response¹¹ in the 293SGlycoDelete cells demonstratesthat the GlycoDelete strategy does not noticeably interfere with therole ofN-glycans in quality control in the endoplasmic reticulum.

Example 2 The GlycoDelete Cell Line is Suitable for Expression ofGlycoproteins with Reduced Heterogeneity and Length of N-glycans,without Affecting Protein Function.

The effect of stable GlycoDelete engineering was assessed on atransiently overexpressed, secreted cytokine (the humangranulocyte/macrophage colony stimulating factor, hGM-CSF¹³), on astably overexpressed GPCR, the 5HT1DR¹² (Example 3), on a transientlyoverexpressed monoclonal antibody (anti-CD20, obinutuzumab)¹⁴ (Example4) and on a transiently overexpressed Fc-containing fusion protein(anti-TNF, etanercept) (Example 5).

Furthermore, GM-CSF was transiently expressed in 293S, 293 SGnTI−/− and293 SGlycoDelete cells and purified from the culture medium. GM-CSFproduced in 293S or 293 SGnTI−/− cells consists of three main glycoforms(corresponding to occupancy of zero, one or two N-glycosylationsites),¹⁵ which are converted to a form of the protein with a lowermolecular weight (MW) by treatment with peptide-N-glycosidase F(PNGaseF), which cleaves the N-glycosidic bond between the asparagineside chain and N-glycans that contain at least the chitobiose core (FIG.7A). The remaining heterogeneity is due to O-glycosylation,¹⁵ asindicated by its partial disappearance upon sialidase digestion. Incontrast, a lower MW range was observed for the GM-CSF purified from293SGlycoDelete cells (FIG. 7A). PNGaseF treatment of the GlycoDeleteGM-CSF did not cause any change in the observed pattern, demonstratingthe absence of chitobiose-core containing N-glycans. Treatment withsialidase caused a shift in the MW of the GlycoDelete GM-CSF, more sothan in the case of GM-CSF from 293S or 293SGnTI−/− cells, indicatingthe presence of more sialic acid residues on GlycoDelete GM-CSF than onthe other forms (FIG. 7A). This conclusion was also supported by glycananalytics, described below (FIGS. 7B, 8 and 9). After digestion withPNGaseF and sialidase, GM-CSF from all three cell lines ran as singlebands with indistinguishable mobility (it should be noted that thesegels do not resolve nonglycosylated proteins from those modified withthe small GlycoDelete N-glycan stumps), supporting the conclusion thatthe differences between GM-CSF from 293S, 293SGnTI(−) and 293SGlycoDelete cells were due to glycosylation differences; this wasconfirmed by mass spectrometric analysis of the intact proteins (FIG.10).

To further characterize the N-glycans on GM-CSF from 293SGlycoDelete and293SGnTI(−) cells, the samples were analyzed by matrix-assisted laserdesorption ionization (MALDI)—mass spectrometry (FIGS. 7B and 8).Analysis of 293S GM-CSF glycans by capillary electrophoresis (FIG. 9)revealed a typical heterogeneous mixture of multibranched complex-typeglycans. The level of sialylation was low, probably owing to the rapidtransfer of the cells to serum-free medium during protein production.The glycopeptide containing N37 of 293SGnTI(−) GM-CSF was detected as aMan5GlcNAc2(Fuc) N-glycosylated peptide (FIG. 7B, top), in agreementwith previous findings.^(4,16) These ions are absent from the spectrumof GM-CSF produced in 293SGlycoDelete cells, in which three newglycopeptide masses were detected. These masses are consistent withN-acetylhexosamine (HexNAc) glycopeptide, Hex-HexNAc glycopeptide andN-acetylneuraminic acid (Neu5Ac)-Hex-HexNAc glycopeptide (FIG. 7B).Similar observations were made for the glycopeptide containing N27 (FIG.8).

To confirm the identity and linkage of the hexose and Neu5Ac units onGlycoDelete GM-C SF glycopeptides, exoglycosidase digests were performedwith an α-2,3-/α-2,6-/α-2,8-sialidase and β-1,4-galactosidase (FIG. 7B).This allowed establishment that the di- and trisaccharide modifiedpeptides are Gal-β-1,4-GlcNAc and Neu5Ac-α-2,3-Gal-β-1,4-GlcNAc,respectively. The presence of these glycans—not just the single GlcNAcendoT digestion product—on proteins produced in the GlycoDelete cellsshows that galactosyltransferases and sialyltransferases in the Golgiact on the GlcNAc stumps generated by endoT. This confirms that endoTdeglycosylation of GM-CSF must have occurred intracellularly and notafter secretion. Quantification of the relative peak intensities of theprotein spectra before and after sialidase treatment indicated that ˜75%of glycans on GM-C SF from GlycoDelete cells were sialylated.

The influence of the GlycoDelete glycan alteration on properties ofGM-CSF was then investigated. A ThermoFluor assay¹⁷ showed that themelting temperatures of GM-CSF from Escherichia coli (nonglycosylated,Tm=58.9±0.6° C.), 293S cells (complex type N-glycosylation, Tm=61.2±3.2°C.) and 293SGlycoDelete cells (Tm=61.5±0.2° C.) were not significantlydifferent (Kruskal-Wallis test, n=4, P>0.05; mean±s.d.) (FIG. 7C).Furthermore, in a TF1 human erythroleukemia cell—proliferation assay¹⁸(FIG. 7D), the bioactivity of GM-CSF from 293S and 293SGlycoDelete cellswas highly similar.

To assess whether GlycoDelete glycans contribute to the antigenicity ofGM-CSF, rabbits were immunized with GM-CSF from 293SGlycoDelete cells.Binding of serum antibodies to undigested, sialidase-treated orsialidase- and galactosidase-treated 293SGlycoDelete GM-CSF wasdetermined by ELISA. GM-CSF from which the GlycoDelete glycan structureshad been removed and GM-CSF with the GlycoDelete glycans present wererecognized equally well, indicating that the GlycoDelete glycans did notform new immunogenic epitopes on GM-CSF in rabbits (FIGS. 7E and 7F).

Example 3 The GlycoDelete Cell Line is Suitable for Stable Expression ofGlycoproteins with Reduced Heterogeneity and Length of N-glycans

To confirm that GlycoDelete is compatible with stable transfection-basedprotein production and that it can process membrane proteins, a stablecell line was generated in which, next to the stable GlycoDeleteengineering, a GPCR, the 5HT1DR, ¹² was stably overexpressed. Treatmentof membrane protein extracts with PNGaseF revealed a large shift in themolecular weight (MW) of the 5HT1DR stably overproduced in 293SGnTI−/−cells. Contrary to this, whether or not treated with PNGaseF, thereceptor produced in 293SGlycoDelete cells ran at approximately the sameMW as PNGaseF-treated receptor from 293SGnTI−/− cells. It was concludedthat, in 293SGlycoDelete cells, ST-endoT completely hydrolyzed the5HT1DR N-glycans (FIG. 11).

Example 4 Antibodies Produced by the GlycoDelete Cell Line have the sameAffinity for their Ligand, but Longer Circulation Times In Vivo

To further explore the scope of GlycoDelete technology, the monoclonalanti-CD20 antibody obinutuzumab (GA101)¹⁴ was transiently expressed in293S and 293SGlycoDelete cells and purified from the cell culturemedium. The cell lines produced similar amounts of the antibody (FIG.12). 293S-produced anti-CD20 carries core-fucosylated biantennaryN-glycans typical of IgGs¹⁹ on its only Fc-linked N-glycosylation site(N297 in the heavy chain Cγ2-domain) (FIG. 13). As expected, treatmentwith PNGaseF reduced the MW (FIG. 14A). In contrast, the heavy chain ofthe antibody produced in 293SGlycoDelete cells ran at approximately thesame MW as the heavy chain of the PNGaseF-treated antibody from 293Scells, and the MW was not further reduced by PNGaseF treatment (FIG.14A). This result is consistent with the N-glycans on this IgG havingbeen cleaved by endoT. Thus, the GlycoDelete cells also process hIgGFc-linked N-glycans.

To further characterize the glycans on 293SGlycoDelete anti-CD20, thedifferent glycoforms of the tryptic IgG peptide containing theN-glycosylation site were quantified using liquidchromatography—electrospray ionization mass spectrometry (LC-MS/MS) inselected reaction monitoring (SRM) mode (FIG. 14B). Furthermore, LC-MSanalysis of the intact antibody was performed with and withoutdissociation of the chains through reduction (FIGS. 15A-15C). TheLC-MS/MS analysis revealed that the GlycoDelete protein was modifiedwith HexNAc, Gal-HexNAc and Neu5Ac-Gal-HexNAc N-glycans, as alsoobserved for GM-CSF. Quantification of the relative glycopeptide peakareas of samples before and after sialidase treatment allowedestablishment that 19% of the anti-CD20 carries the sialylatedtrisaccharide and 72% carries the Gal-GlcNAc disaccharide, the remainderbeing the GlcNAc-modified peptide. In SRM-mode LC-MS/MS peptideanalysis, the Hex5-HexNAc2 glycopeptide that is dominant in 293 SGnTI(−)IgG was below the detection limit for 293SGlycoDelete IgG. Intactprotein LC-MS analysis revealed a very small remaining fraction of theHex5-HexNAc2 glycoform, both in 293S- and 293SGlycoDelete-producedantibodies. The amount of Hex5HexNAc2 in both preparations wasquantified at 2.5% of the total glycan pool by DNA-sequencercarbohydrate electrophoresis of a 1:1 mixture of both antibodies (datanot shown).

In addition, flow cytometric analysis of binding to CD20+ cells showedthat GlycoDelete anti-CD20 antigen binding was identical to that of 293Santi-CD20 (FIG. 14C), demonstrating that the antigen-binding fold isunaffected.

As N-glycans make up part of the fold packing contacts in the Cγ2domain, size reduction of these glycans is expected to lead to a drop inTm. Accordingly, the Tm for Cγ2 is ˜64° C. for complex-typeN-glycosylated 293S anti-CD20 and 57° C. for 293SGlycoDelete anti-CD20,similar to the Tm for PNGaseF-digested 293S anti-CD20 (FIG. 14D). Noevidence was found of aggregation of anti-CD20 expressed by 293S or293SGlycoDelete cells by gel filtration chromatography (FIG. 16).

Glycosylation on heavy chain N297 has a major influence on the affinityof binding of antibodies to Fc-γ receptors (FcγRs)²⁰, so the binding of293S and 293SGlycoDelete anti-CD20 to different human FcγRs wasassessed. Surface plasmon resonance experiments (Table 1) showed thatthe human and mouse neonatal FcRs (FcRns) have similar affinities forboth anti-CD20 glycoforms. This is expected because the FcRn bindingsite is not located near the Cγ2 N-glycan site (Roopenian et al., 2007).A competition ELISA was set up for FcγRI, FcγRIIa and FcγRIIb in whichthe anti-CD20 antibodies compete in solution for FcγR binding with aprecoated IgG. In all three cases, a >10-fold reduction in bindingcompetition by 293SGlycoDelete anti-CD20 compared to the 293S anti-CD20was detected (FIG. 14E). FcRIIIa binding affinity, as assessed bybiolayer interferometry (Table 1), was 5.8 times lower for293SGlycoDelete anti-CD20 than for 293S anti-CD20. Similarly, in anantibody-dependent cell-cytotoxicity (ADCC) assay using natural killer(NK) cells as effectors (FIG. 14E), it was found that the half-maximaleffective concentration (EC50) of the specific lysis with293SGlycoDelete anti-CD20 is 6.6 times higher than with 293S anti-CD20.Overall, the GlycoDelete glycosylation of human IgG1 Fc leads to reducedbinding to FcγRs; in the context of producing neutralizing antibodies,this might be desirable to improve safety (Lux et al., 2013).

TABLE 1 Dissociation constants (K_(D)) of the Fc binding with hFcRn,mFcRn and FcγRIIIaV K_(D) fold GlycoDelete 293S reduction hFcRn 6.72E−095.60E−09 0.83 mFcRn 2.24E−10 2.58E−10 1.15 FcγRIIIaV 2.90E−06 5.00E−075.8

Both hFcRn and mFcRn binding was determined with SPR and a K_(D) forboth glycoforms were found within the same range. IgG binding toFcγRIIIaV was determined using BLI. KD of 293SGlycoDelete cells isreduced with a factor 5.8 compared to the WT glycoform.

To assess whether GlycoDelete glycans on the IgG are immunogenic, asimilar immunization experiment as for GM-CSF was performed (FIG. 14F)and concluded that GlycoDelete glycans do not substantially contributeto antigenicity of the anti-CD20 molecule.

Remarkably, pharmacokinetics analysis in mice showed that the initialrapid clearing phase (1 hour post-injection) removed substantially lessof the GlycoDelete anti-CD20 from circulation, leading to doubledlong-term circulation levels. Both glycoforms were subsequently clearedat an equivalent (slow) rate (FIGS. 14G and 17), as was anticipated fromtheir similar FcRn affinity. Owing to the initially higher level, it,therefore, would take 10-12 days longer for the concentration of theGlycoDelete antibody to drop below a required therapeutic thresholdconcentration. This means that considerably higher levels of theGlycoDelete anti-CD20 remained in vivo for a much more prolonged timeperiod. A possible mechanism is that higher sialylation leads to lowerclearance through reduced binding to liver and macrophage lectinreceptors. Potentially, the sialylation levels of GlycoDelete IgG couldbe further enhanced, and this observation suggests that GlycoDelete IgGsmight allow a reduced frequency of dosing for neutralizing therapeuticIgGs that often require long circulation periods in the blood.

Example 5 Chimeric Fc-Containing Molecules Produced by the GlycoDeleteCell Line also have more Homogenous Glycosylation Patterns

Next, etanercept, a recombinant fusion protein consisting of the humantype 2 TNF receptor fused to the constant end of the IgG1 antibody, wastransiently expressed and purified in GlycoDelete cells. Similar to theproteins tested in Examples 2 and 4, LC-MS analysis revealed that the Fcpart of the GlycoDelete protein was modified with HexNAc, Gal-HexNAc andNeu5Ac-Gal-HexNAc N-glycans (FIG. 18). (This was evaluated with theEQQYNSTYR peptide (SEQ ID NO:1) from the Fc chain.)

Subsequent sialidase and galactosidase digest further confirmed theidentity of these sugar groups (FIG. 18). Quantification of the relativeglycopeptide peak areas of samples before and after sialidase andgalactosidase treatment allowed establishment that 25% of etanerceptproduced in these cells has an Fc chain carrying the sialylatedtrisaccharide and 68% carries the Gal-GlcNAc disaccharide, the remainderbeing the GlcNAc-modified peptide (Table 2). These percentages are ingood agreement with those observed for the anti-CD20 antibody,indicating that the glycosylation of Fc chains in the cells is quiteuniform.

TABLE 2 LC-MS area under the curve of the Fc-glycopeptide on etanerceptin three replicated measurements (one of which is shown in FIG. 18):Area under the curve Pep- Pep-GlcNAc- Pep-GlcNAc- Pep- Pep GlcNAc GalGal-Sial Man5 peptide 2044 22361 267000 19712 36 2127 18806 243015 2082310 1865 16690 228098 18072 15 avg 2012 19286 246038 19536 20 Peptide +1640 13989 257195 15 15 sialidase 1051 13234 233126 46 20 4608 14247236880 76 10 avg 2433 13823 242400 46 15 Peptide + 6752 373558 22399 1836 sialidase + 7191 368247 21963 20 10 galactosidase 6640 355005 2012010 15 avg 6861 365603 21494 16 20

In the Table, it can be seen that only trace amounts of the originalmannose-5 glycan are found back in the etanercept sample. 25% of the Fcchain carries the GlcNAc-galactose-sialic acid trisaccharide, 68%carries the GlcNAC-galactose disaccharide and the remainder is singleGlcNAc glycosylated.

Conclusion

In conclusion, this study introduces the GlycoDelete glycoengineeringstrategy as an approach to solving the issue of N-glycosylationheterogeneity in mammalian cell-based glycoprotein production.GlycoDelete involves the optional, but particularly envisaged,inactivation of a single glycosyltransferase (GnTI, encoded by the geneMGAT1) and overexpression of a deglycosylating enzyme, followed bylectin selection. GlycoDelete cells produce proteins with the Gal-GlcNAcdisaccharide or its α-2,3-sialylated trisaccharide derivative and someof the monosaccharide intermediate. This is in contrast to the dozens ofglycan structures produced by wild-type mammalian cells. The GlycoDeletestrategy strikes a balance between retaining the folding-enhancingfunctions of N-glycans and avoiding the extensive heterogeneityintroduced through mammalian Golgi N-glycan processing. In addition tothe advantages of reduced N-glycan complexity in biopharmaceuticalmanufacturing, examples of the therapeutic benefit of similar short,simple N-glycans generated in vitro have been reported.^(21,23)Furthermore, it has been shown that GlycoDelete engineering favorablyalters the characteristics of antibodies when the therapeutic goal isantigen neutralization without the need for additional effectorfunction. Therefore, GlycoDelete could lead to “biobetters,” an area ofinterest in the biopharmaceutical industry.²⁸ The strategy appears to beparticularly well suited for expression of Fc-containing molecules,since it prolongs circulating half-life just by altering theglycosylation of the conserved N297 residue. This has importanttherapeutic advantages for, e.g., therapeutic IgG injections, which canbe done much less frequently (e.g., half as frequently) while retainingthe same efficacy because of the same affinity for the ligand.

Material and Methods General Cell Culture and Transfection.

293SGnTI(−) cells were maintained in a humidified incubator at 37° C.and 5% CO2 in DMEM/F12 (Gibco) with 10% FBS, 292 μg/mL L-glutamine, 100units/mL penicillin and 100 μg/mL streptomycin (all Sigma-Aldrich).

For small-scale transfections, the cells were plated in a six-well plate48 hours before transfection at ˜150,000 cells per well. They weretransfected using the TransIT-293 Transfection Reagent (Mirus Bio LLC)according to the manufacturer's instructions. For transient orlarge-scale transfections, cells were transfected with the calciumphosphate transfection method. Raji cells were cultured in RPMI 1640+10%FBS+2 mM L-Glutamine.

All cell lines were routinely tested for mycoplasma contamination withthe Plasmotest kit (InvivoGen).

Transient EndoT Expression.

The endoT fusion constructs (pCAGGS-GM2S-endoT and pCAGGS-ST-endoT) andthe secreted endoT construct (pCAGGS-s-endoT) were transientlytransfected to 293SGnTI(−) cells as described above. Supernatant andcell lysate samples were analyzed to assess targeting domain performance(FIG. 2).

In Vivo De-N-Glycosylation by Transient Transfection of EndoT-Fusions

De-N-glycosylation by endoT was evaluated by transfecting all endoTconstructs to 293SGnTI(−) cells stably and inducibly expressing the Flt3receptor extracellular domain (FIGS. 3A and 3B).

Construction of the Plasmid for Stable ST-EndoT Expression(pcDNA3.1(-)Zeo-ST-endoT).

The ST-endoT PCR fragment was cloned into a pCR®II-TOPO® plasmid (LifeTechnologies). The resulting Topo-ST-endoT plasmid (reverse complementinsertion) was digested with XhoI and KpnI and the insert was purified.The pcDNA3.1/zeo(−) plasmid was digested once with XhoI and PvuI, andonce with PvuI and KpnI and then a 1.5 kb and a 3.6 kb fragment werepurified, respectively. A subsequent three-point ligation with thevector fragments and the ST-endoT fragment resulted in thepcDNA3.1/zeo-ST-endoT plasmid.

TABLE 3 Primer sequences for ST-endoT cloning Oligo Sequence PR15′-AACAAGGACGTACCCGTTAAAGAACTGCA-3′ (SEQ ID NO: 2) PR25′-CGCGAGCACCGTACCCGTTAAAGAACTGCA-3′ (SEQ ID NO: 3) PR35′-CTCGAGATGGTACCCGTTAAAGAACTCXAGTTGAGAGC-3′ (SEQ ID NO : 4) PR45′-GCACCTGAGGTTACAGATCTTCTTCAGAAATAAGCTTTTGTTCAGCGTTAACCATAGCGTAGTAGTTGATGG-3′ (SEQ ID NO: 5) PR55′-GCACTCGAGATGATTCACACCAACCTGAAGA-3′ (SEQ ID NO: 6) PR65′-TTAACGGGTACGTCCTTGTTCCACACCTG-3′ (SEQ ID NO: 7) PR75′-GCACTCGAGATGTGGCTGGGCCGCCGGG-3′ (SEQ ID NO: 8) PR85′-TTAACGGGTACGGTGCTCGCGTACAGGAGCC-3′ (SEQ ID NO: 9) PR95′-TCGAGATGAAGACTATCATTGCTTTGAGCTACATTTTCTGTCTGGTTTGG GCCCAAGACGTAC-3′(SEQ ID NO: 10) PR105′-GTCTTGGGCCCAAACCAGACAGAAAATGTAGCTCAAAGCAATGATAGTC TTCATC-3′(SEQ ID NO: 11) PR11 5′-GTGCTGCTCCTGGTTCTTTC-3′ (SEQ ID NO: 12) PR125′-TCAGCCATAGAACCGAAACC-3′ (SEQ ID NO: 13) PR135′-CTAGAATTCGCGATATCCCGGGCCCAGCGCTGCGGCCGCTCGAGCTAGC GTTTAAACT-3′(SEQ ID NO: 14) PR145′-GATCAGTTTAAACGCTAGCTCGAGCGGCCGCAGCGCTGGGCCCGGGATA TCGCGAATT-3′(SEQ ID NO: 15) PR15 5′-GCAGTCGACCATGTCCCCACTGAACCAGTCAGC-3′(SEQ ID NO: 16) PR16 5′-GCAGCGGCCGCGGAGGCCTTCCGGAAAGGGAC-3′(SEQ ID NO: 17) PR175′-AAACTTAGGCGGGAGCCACCTGGCTGGTCTCAGTACTGGCCTTCCGGAA AGGGAC-3′(SEQ ID NO: 18) PR18 5′-CTCCCGCCTAAGTTTAAACGTTTAACCCGGGTAAATTCCGC-3′(SEQ ID NO: 19) PR19 5′-GATTATGATCAGTTTAAACACTAGTAAATTCTAGAGTCGCGGC-3′(SEQ ID NO: 20) PR20 5′-CTCAAGGGCCCCTTGACC-3′ (SEQ ID NO: 21) PR215′-CGAGCAGAATTCAATGGTGATGATGGTGATGCTCCTGGACTGGCTCCCA G-3′(SEQ ID NO: 22)

Stable Cell-Line Generation

293SGnTI(−) cells were transfected in a small-scale transfection withpcDNA3.1(−)Zeo-ST-endoT. Selection was initiated with 15 μg/mL ConA 48hours after transfection. After 14 days, the cells were trypsinized andreplated in conditioned medium (medium of 2-day-old 293SGnTI(−)cultures, sterile filtered and mixed with 50% (v/v) fresh DMEM/F12)containing 10 μg/mL ConA. After 14 days, five large and nicely separatedcolonies were picked and expanded in the presence of 10 μg/mL ConA. Thetwo fastest growing clones were further analyzed.

293SGnTI(−) and 293SGlycoDelete Growth Curve

Cells from a 70-80% confluent culture were first diluted to ˜60,000cells per milliliter, counted again (time point 0 hour) and transferredto a six-well plate (180,000 cells per well). At each time point, threewells were detached by pipetting up and down the medium, and the viablecells were counted for each well using trypan blue exclusion and ahemocytometer. The result shown in FIG. 1C represents one of tworeplicate experiments.

Gene-Expression Analysis.

RNA isolation and sample preparation for analysis on GeneChip Human Exon1.0 ST Arrays (Affymetrix) were as follows.

Total RNA was extracted from three replicates cultures of both lineswith the RNEASY® Midi kit (Qiagen), according to the manufacturer'sinstructions. RNA quality was assessed on a 2100 BIOANALYZER® using RNA6000 Pico chips(Agilent Technologies, Santa Clara, Calif., USA). Allsamples had an RNA Integrity Number (RIN) of 9.5 or better. Afterspiking the total RNA samples (RNA sample preparation, see OnlineMethods) with bacterial poly-A RNA positive controls (Affymetrix, SantaClara, Calif., USA), every sample was reverse transcribed, converted todouble-stranded cDNA, in vitro transcribed and amplified using theAMBION® WT Expression Kit. The obtained single-stranded cDNA wasbiotinylated after fragmentation with the WT Terminal Labeling kit(Affymetrix), according to the manufacturer's instructions. Theresulting samples were mixed with hybridization controls (Affymetrix)and hybridized on GENECHIP® Human Exon 1.0 ST Arrays (Affymetrix). Thearrays were stained and washed in a GENECHIP® Fluidics Station 450(Affymetrix), and scanned for raw probe signal intensities with theGENECHIP® Scanner 3000 (Affymetrix). Exon array data are MIAME compliantand available from the ArrayExpress database (on the World Wide Web atebi.ac.uk/arrayexpress) under accession number E-MEXP-3516.

A combination of the R Statistical Software Package (on the World WideWeb at r-project.org) and Affymetrix Power Tools (APT; Affymetrix) wereused for the quality control and differential expression analysis of theexon array data, partly as described earlier.⁷ Briefly, exon- andgene-level intensity estimates were generated by background correction,normalization and probe summarization using the Robust Multi-arrayAverage (RMA) algorithm with APT. Quality control of the data before andafter normalization was performed in R through the generation of variousplots such as box and density plots. Genes of which the expression wasundetected in both lines were excluded from further analysis. A gene wasconsidered to be detected when more than half of its exons were detectedabove the background (p<0.05) in at least two of the three biologicalreplicates of that cell line. Genes of which the expression was belowthe estimated noise level in both lines were also removed from furtheranalysis. The noise level threshold was set at the signal intensitylevel (the APT output intensity, averaged over the three replicates),which eliminated “detection” of expression of more than 95% of the geneson the Y-chromosome, which is absent from the 293 lineage (which wasderived from a female embryo) and thus serves as an appropriate internalnegative control.

Differential gene expression analysis was performed using a linear modelfit implemented in the R Bioconductor package Limma,⁸ considering onlycore probesets. The Benjamini-Hochberg (BH) method was applied tocorrect for multiple testing.

GM-CSF Production and Purification.

The plasmid for transient GM-CSF expression (pORF-hGM-CSF-6xHis) wastransiently transfected to both 293SGnTI(−) and 293SGlycoDelete celllines. The secreted GM-CSF was purified from the medium.

Construction of the pORF-hGM-CSF-6xHis plasmid. A partial CDS of thehuman GM-CSF C-terminally tagged with six His residues was amplifiedwith primers PR18 and PR19 from the pORF-hGM-CSF plasmid (Invivogen, CA,USA). The PCR fragment and the pORF-hGM-CSF plasmid were digested withApal and EcoRI and ligated both fragments to result in thepORF-hGM-CSF-6xHis plasmid.

Human GM-CSF purification. 293SGnTI−/− and 293SGlycoDelete cells weretransiently transfected with the pORF-hGM-CSF-6xHis plasmid (transienttransfection, see online methods). Four days post-transfection, 50 ml ofmedium containing the expressed protein was harvested and dialyzedagainst buffer A (20 mM NaH₂PO₄, 0.5 M NaCl and 20 mM imidazole pH 7.5)using 3 kDa MWCO membranes. The dialysate was loaded onto a 1 mlHis-Trap HP column charged with Ni²⁺ ions (GE Healthcare UK Ltd,Buckinghamshire, UK). Then, the column was washed with buffer A untilthe A280 had dropped back to the baseline. After washing the column withten-column volumes 6% buffer B (20 mM NaH₂PO₄ pH 7.50+20 mM NaCl+0.5 Mimidazole), bound proteins were eluted with 100% buffer B and collectedin 1 ml fractions. The presence of GM-CSF in the collected fractions wasverified by tricine SDS-PAGE gel electrophoresis.⁹ The proteinconcentration was measured based on the A₂₈₀ absorbance of theGM-CSF-containing fractions versus buffer B as a blank. Concentrationswere calculated using the theoretical absorption coefficient with allcysteine residues in disulfide linkages (13980 M⁻¹ cm⁻¹), as calculatedby the protparam tool (on the World Wide Web atweb.expasy.org/protparam).¹⁰

Anti-CD20 Production and Purification.

Anti-CD20 was transiently expressed in both 293S and 293SGlycoDeletecell lines as described above and purified as follows: 4 dayspost-transient transfection of 293S and 293SGlycoDelete cells with thevector containing anti-CD20 (transient transfection, see onlinemethods), the medium containing the expressed protein was harvested andloaded onto an affinity column 5 ml HITRAP® Mab Select SuRe (GEHealthcare UK Ltd, Buckinghamshire, UK). The column was then washed withPBS until A₂₈₀ had dropped back to baseline. Bound proteins were elutedwith 50 mM glycine pH 3.5 and collected in 1 ml fractions. The presenceof anti-CD20 in the collected fractions was verified by tricine SDS-PAGEgel electrophoresis. A buffer exchange was performed on the pooledfractions that contained anti-CD20 to a 25 mM histidine 125 mM NaClbuffer at pH 6.0. Antibody concentration in the purified samples wasmeasured with a Synergy MX spectrophotometer (Biotek, VT, USA). Theprotein concentration was measured based on the A₂₈₀ absorbance of thepurified antibody. Concentrations were calculated using the theoreticalextinction coefficient.

5HT1D receptor expression and sample preparation. Detailed methods forstable 5HT1DR-expressing cell line generation, 5HT1D sample preparationand analysis are as follows.

Construction of the pT-REx-5HT1DRho and pT-REx-5HT1DRho-IRESdsRed2plasmid. The pT-REx-DEST30 plasmid (Invitrogen) was amplified in adam/dcm methylation-deficient E. coli strain and digested with BclI andXbaI. A dsDNA insert was created by annealing oligos PR11 and PR12.Subsequent ligation of the dsDNA insert into the XbaI/BclI-digestedpT-REx-DEST30 fragment generated the pT-REx-MCS plasmid.

The CDS for the 5-hydroxy tryptamine 1D receptor (NM_00864) from a humanfetal brain cDNA library was amplified using primers PR13 and PR14 andcloned into a pCR®II-TOPO® plasmid (Invitrogen), generating theTopo-5HT1D plasmid. A Rho1D4-tagged 5HT1DR fragment was amplified fromthe Topo-5HT1D plasmid with primers PR13 and PR15. The PCR fragment wasdigested with SalI and the pT-REx-MCS plasmid with PmeI and SalI,followed by dephosphorylation. These fragments were ligated to result inthe pT-REx-5HT1DRho plasmid.

The IRESdsRed2 fragment from the pLV-tTR/KRAB-Red plasmid (a kind giftof Prof. Peter Vandenabeele, VIB-UGhent) was amplified with primers PR16and PR17. The pT-REx-5HT1DRho plasmid was digested with Pmel and usedwith the IRESdsRed2 fragment in a cloneEZ (GenScript USA Inc., NJ, USA)reaction. This resulted in the pT-REx-5HT1DRho-IRESdsRed2 plasmid.

5HT1DR expressing 293SGnTI−/− and 293SGlycoDelete clones. Cell lineswere generated, stably and inducibly expressing the 5HT1D receptor bytransfecting 293SGnTI−/− with the pT-RExL-5HT1DRho-IRESdsRed2 plasmidand 293SGlycoDelete cells with pTRExL-5HT1DRho orpT-RExL-5HT1DRho-IRESdsRed2. Selection was performed with G418(Sigma-Aldrich) at 600 μg/ml (293SGnTI−/− cells) and at 150 μg/ml G418(293SGlycoDelete cells). The G418-resistant cells were then subjected tolimiting dilution cloning in conditioned medium. Expression of the 5HT1Dreceptor was induced with 2 μg/ml tetracycline and 1 mM valproate(Sigma-Aldrich). The 293SGnTI−/− 5HT1DR clone expressing the highestintensity of red fluorescence was selected after 2-3 days of inductionby fluorescence microscopy.

ELISA analysis for 5HT1DR expression in 293SGlycoDelete clones. ForELISA analysis of the 5HT1DR-expressing 293SGlycoDelete clones, cellswere collected from 24-well plates after 2-3 days induction with 2 μg/mltetracycline and 1 mM valproate (Sigma-Aldrich). The cells were spundown and the supernatant discarded. Cells were lysed with RIPAbuffer+protease inhibitors by incubating for 20 minutes on ice. Thedebris was removed by spinning down the samples at 12,000 rpm for 10minutes. Protein was determined in a bicinchoninic acid (BCA) assay(Pierce Biotechnology Inc., Rockford, Ill., USA) according to themanufacturer's instructions. 15 μg of each sample, of a positive controlsample of 5HT1DR produced in P. pastoris and of a293SGlycoDelete-negative control sample were coated overnight at 4° C.on a maxisorb plate. The plate was washed three times with water and onetime with wash buffer (PBS+0.1% TWEEN®−80). Blocking buffer (PBS+1% milkpowder) was added to each well and incubated for 2 hours at roomtemperature. After washing, the anti-rho1D4 antibody (University ofBritish Columbia, Vancouver, Canada), diluted 1/100 in sample buffer(PBS+0.05% TWEEN®+0.5% milk powder) was added and the samples wereincubated for 1 hour at room temperature. The plate was again washed andthen an anti-mouse IgG coupled to HRP secondary antibody (GE HealthcareBiosciences, Pittsburgh, Pa., USA) and diluted 1/5000 in sample bufferwas added to the samples. Finally, the plate was again washed andsamples were analyzed with the BD OptEIA™ TMB substrate reagent set (BD,Franklin Lakes, N.J., USA), according to the manufacturer'sinstructions.

5HT1D receptor expression and sample preparation. 293SGnTI−/− and293SGlycoDelete cell lines were generated, stably and induciblyexpressing the 5HT1D receptor. Detailed methods for the generation of5HT1DR expression constructs and subsequent generation of stable5HT1DR-expressing clones are described in Supplementary Note 1. Theselected 5HT1DR-expressing clone of each line was induced with 2 μg/mltetracycline and 1 mM valproate. Three days post-induction, cells werecollected. Cell pellets were resuspended in 5 ml of 20 mM Tris-HCl pH8.0+1 mM EDTA+Complete EDTA-free protease inhibitors (Roche, Mannheim,Germany). 1.25 ml of each sample was sonicated on ice (15 cycles, eachcycle: 1 second on and 5 seconds off, at 20% amplitude) with a VCX500sonicator (Sonics & Materials Inc., Newtown, Conn., USA). The lysateswere immediately centrifuged for 10 minutes at 13,000 rpm and 4° C. andsolubilized the pellets in the buffer described above +0.35 mM NaCl and0.5% n-dodecyl-β-D-maltoside. Debris was removed by immediatelycentrifuging samples again for 10 minutes at 13,000 rpm at 4° C.

To assess the presence of PNGaseF-sensitive N-glycans on the 5HT1Dreceptor, 50 μl aliquots of the samples, supplemented with 1% IgepalCA-630 and 200 U of PNGaseF (in-house production), or no enzyme, wereincubated overnight at 37° C. The samples were analyzed byimmunoblotting using a mouse anti-rho1D4 primary antibody (University ofBritish Columbia, Vancouver, Canada), diluted 1/250.

Sialidase, Galactosidase and PNGaseF Digests and SDS-PAGE.

The glycoproteins were diluted in 50 mM of phosphate buffer (pH 7.0)containing 40 mM of β-mercaptoethanol and 0.5% SDS. Samples wereincubated for 10 minutes at 98° C. After cooling, 1% Igepal CA630 andthe appropriate enzymes were added: 100 U of PNGaseF (producedin-house), 200 mU of Arthrobacter ureafaciens sialidase (producedin-house), 2 mU of Streptococcus pneumoniae β-1,4-galactosidase(Prozyme) or combinations. The samples were incubated overnight at 37°C. and analyzed the following day on a tricine SD S-PAGE gel.

Thermofluor assays. Thermofluor assays were performed as described inEricsson et al.¹⁷ Briefly, purified protein was diluted to anappropriate assay volume (10-20 μl) in a solution containing buffer (PBSfor GM-CSF and His buffer—25 mM histidine, 125 mM NaCl, pH 6.00—foranti-CD20) and 20× concentrated Sypro orange dye (5000× solution inDMSO, Life Technologies, Paisley, UK). Each experiment was run as atechnical triplicate, and triplicate blank measurements with no testprotein were included. Fluorescence in function of temperature wasrecorded in a 348-well LIGHTCYCLER® 480 (Roche, Basel, Switzerland) from25° C. to 95° C. with a temperature ramping rate of 0.01° C.second.

Before any calculations and statistical analyses, datasets with obvioustechnical problems (abnormally high initial fluorescence, off-scalefluorescence) were omitted entirely. Melting temperatures werecalculated as the V₅₀ value of a Boltzmann sigmoidal curve fitted to theaveraged data points of the three replicates in each experiment. For thecurve fitting procedure, data points beyond the maximal fluorescencewere omitted. When more than one melting point was calculated from asingle experiment, an appropriate subset of data points, including theminimal and maximal fluorescence values at temperatures just below andabove that melting point, was used. For graphing, the raw datasets wereaveraged, blank (averaged) corrected and then normalized (minimalvalue=0%, maximal value=100%).

For the GM-CSF samples, an average T_(m) was calculated from a set ofindependent experiments (E. coli: n=4, 293S: n=3, 293SGlycoDelete: n=3).Tests were run to determine whether the average T_(m) ^(S) werestatistically significantly different by Kruskal-Wallis one-way ANOVA(P=0.05) and Dunn test for multiple comparisons (α=0.05).

MALDI Glycopeptide Analysis.

GM-CSF of the different cell lines (1-4 μg of protein in 20 μL) wassupplemented with 10 μL of 3× tricine gel loading buffer (1.5 MTris-HCl, pH 8.45, 35% glycerol, 10% SDS, 0.01% Coomassie and 30 mM DTT)and incubated for 10 minutes at 98° C. 3 μL of a 500 mM iodoacetamidestock was added, and the samples were incubated for 1 hour in the dark.The samples were separated on a 12% tricine SDS-PAGE gel and cut out thebands.

Detailed methods for in-gel tryptic digestion are as follows. Gel pieceswere washed three times with 50% acetonitrile (ACN), dried with 100% ACNand allowed to reswell in 100 mM NH₄HCO₃. Gel pieces were further driedin a SPEEDVAC®. 750 ng of trypsin (Promega, Madison, Wis., USA) wasadded and the gel pieces were allowed to reswell for 5 minutes. 100 mMNH₄HCO₃ was added to cover all gel pieces and the vials were incubatedovernight at 37° C. 50 μl 100 mM NH₄HCO₃ was added to each vial and thesamples were incubated on a shaker for 15 minutes. 50 μl 100% ACN wasadded and vials were incubated on a shaker for 15 minutes. Supernatantswere collected in fresh vials. 50 μl 5% formic acid in 50% ACN was addedand vials were incubated for 15 minutes on a shaker. The supernatantswere collected. The 5% formic acid step was repeated once. Supernatantswere pooled per sample and dried in a SPEEDVAC®, then reconstituted with20 μl 50 mM phosphate buffer, pH 7.0 and 1 mM Pefabloc (Sigma-Aldrich).

The tryptic peptides were treated with either no enzyme, 50 mU ofα-2,3-sialidase (Takara Bio Inc.), or 200 mU A. ureafaciens sialidaseand 2 mU of Streptococcus pneumoniae β-1,4-galactosidase (Prozyme). Alldigests were incubated for 24 hours at 37° C., dried in a SPEEDVAC®,reconstituted with 10 μL of 0.2% trifluoroacetic acid (TFA)(Sigma-Aldrich) and cleaned up with C18 ZIPTIP® pipette tips (Millipore)according to the manufacturer's instructions. Samples were analyzed with6-aza-2-thiothymine (ATT) matrix saturated in 50% acetonitrilecontaining 0.1% TFA, on a 4800 MALDI TOF/TOF Analyzer (AppliedBiosystems) in the positive ion mode. The reported m/z values wereobserved in several iterations of technical optimizations and theresults of the fully optimized experiments are shown.

LC-MS/MS Glycopeptide Analysis.

9 μg anti-CD20 were diluted in 20 μL of 50 mM phosphate buffer, pH 7.0.Either no enzyme, 100 mU of Arthrobacter ureafaciens sialidase (producedin-house) or 2 mU of β-1,4-galactosidase (Streptococcus pneumoniae) and100 mU of sialidase were added, and the mixture was incubated for 4hours at 37° C. The samples were denatured in a 2 M urea, 10 mM DTT, 50mM ammonium bicarbonate buffer for 30 minutes at 60° C. Iodoacetamidewas added to a concentration of 20 mM and the samples were incubated inthe dark for 30 minutes. Next, the samples were digested with 1/50 (w/w)trypsin (Promega) and incubated overnight at 37° C.

The samples were loaded directly on an Acclaim PEPMAP™ 100 analyticalcolumn (L×ID 15 cm×75 μm, C18, 3 μm, 100 Å) (Thermo) at a flow rate of300 nL per minute, on a U3000-RSLC system (Thermo). Mobile phases were0.1% HCOOH in H2O (solvent A) and 0.1% HCOOH in acetonitrile (ACN)(solvent B). The samples were separated with a 30-minute gradient,ranging from 2% to 40% solvent B, and the eluting peptides were sprayeddirectly into a 4000 Q TRAP® mass spectrometer (AB Sciex) with theNANOSPRAY® II ESI source (AB Sciex). A selected-reaction-monitoring(SRM) method was used to target the glycosylated peptide EEQYNSTYR,where the triple quadrupole cycled through the following SRM transitionlist with a dwell time of 250 ms: Pep-GlcNAc: 696.8 (2+)/526.3 (+) and696.8 (2+)/1189.5 (+) (DP 81.9 V, CE 39.8 eV), Pep-GlcNAc-Gal: 777.8(2+)/526.3 (+) and 777.8 (2+)/1,189.5 (+) (DP 87.8 V, CE 43.9 eV),Pep-GlcNAc-Gal-Sial: 923.4 (2+)/526.3 (+) and 923.4 (2+)/1,189.5 (+) (DP98.4 V, CE 51.2 eV). The 526.3-Da fragment ion (y4-ion, STYR) was usedas quantifier, and the 1,189.5-Da fragment ion (loss ofsugar-modification group) was used as qualifier. The analysis andprocessing of the data was done with Skyline.²⁵ This experiment wasperformed two times. One of the experiments was conducted as a technicalduplicate, the other one as a technical triplicate.

Ratio of Sialylated and Galactosylated Glycans.

To calculate the percentage of GlycoDelete glycans that are sialylated,the area under the peak was extracted from the MALDI MS spectra for theGal-GlcNAc-N (m/z=3622.3) and GlcNAc-N (m/z=3460.2) glycopeptides ofboth the undigested (A_(GalGlcNAcUndig) and A_(GlcNAcUndig)) andα-2,3-sialidase digested (A_(GalGlcNAcDig) and A_(GlcNAcDig))GlycoDelete GM-CSF samples. The percentage of sialylated glycans wascalculated as shown in the formula below. Gal-GlcNAc-N peak areas werefirst normalized to GlcNAc-N peak areas in both spectra. The resultingvalue for the Gal-GlcNAc-N peak from the undigested sample wassubtracted from the value for the Gal-GlcNAc-N peak from thesialidase-digested sample. Then, this difference was divided by thesummed normalized peak areas of the GlcNAc and GalGlcNAc peaks in thedigested sample (total normalized peak area of N27 or N37 encompassingglycopeptides).

${\% \mspace{14mu} {sialylated}\mspace{14mu} {glycans}} = {\frac{\left\lbrack \frac{A_{GalGlcNAcDig}}{A_{GlcNAcDig}} \right\rbrack - \left\lbrack \frac{A_{GalGlcNAcUnDig}}{A_{GlcNAcUnDig}} \right\rbrack}{\left\lbrack \frac{A_{GlcNAcDig}}{A_{GlcNAcDig}} \right\rbrack + \left\lbrack \frac{A_{GalGlcNAcDig}}{A_{GlcNAcDig}} \right\rbrack}*100\%}$

To calculate the percentage of GlycoDelete glycans that aregalactosylated (disaccharide), the same datasets were utilized. Thepercentage of galactosylated glycans was calculated as shown in theformula below. Peak areas for Gal-GlcNAc-N were again first normalizedin both the sialidase-digested and undigested samples. The normalizedpeak area for the undigested Gal-GlcNAc-N peak was then divided by thesummed normalized peak areas of the GlcNAc-N and Gal-GlcNAc-N peaks inthe digested sample (the total normalized peak area of N27 or N37encompassing glycopeptides).

${\% \mspace{14mu} {galactosylated}\mspace{14mu} {glycans}} = {\frac{\left\lbrack \frac{A_{GalGlcNAcUnDig}}{A_{GlcNAcUnDig}} \right\rbrack}{\left\lbrack \frac{A_{GlcNAcDig}}{A_{GlcNAcDig}} \right\rbrack + \left\lbrack \frac{A_{GalGlcNAcDig}}{A_{GlcNAcDig}} \right\rbrack}*100\%}$

GM-CSF Bioactivity Experiments and TF1 Proliferation Assay.

TF1 cells (ATCC No. CRL-2003) were maintained in RPMI 1640, 10% (v/v)FBS, 2 mM of L-Gln and 2 ng/mL of recombinant human GM-CSF at 27° C., 5%CO2. Before starting the assay, cells were washed three times withmedium without cytokines. The cells were subsequently put back in medium(200,000 cells per milliliter) without cytokines and left for 2 hours at37° C.

Upon initiation of the assay, cells were plated in a 96-well plate(20,000 cells per well in 100 μL medium) and serial dilutions (54 ng/mLto 8 pg/mL) of the different glycoforms of GM-CSF were added. Cells wereincubated for 48 hours, 72 hours and 96 hours before performing the MTTassay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) asdescribed.²⁸ Briefly, 20 μl of MTT (5 mg/mL stock) was added per welland incubated. After 4 hours at 37° C., 80 μL of stop solution (10% SDSin 0.01 M HCl) was added, and the plate was further incubated overnightat 37° C. Finally, optical density was measured at 595 nm. The datapoints plotted in FIG. 2 represent mean values from three technicalreplicates. The error bars are s.d. The reported differences between theGM-CSF glycoforms were observed in several iterations of technicaloptimization of these experiments. The results of the fully optimizedbioactivity experiment are shown.

Rabbit Immunizations.

New Zealand White male or female rabbits, aged 13-16 weeks (two rabbitsfor each antigen, results from only one rabbit shown in FIGS. 7A-7F and14A-14G) were injected with 293S GM-CSF, GlycoDelete GM-CSF, 293Santi-CD20 or GlycoDelete anti-CD20. 50 μg of antigen in 500 μL ofantigen solution (50 μg of protein diluted in 0.9% NaCl solution up to500 μL)+500 μL of complete Freund's adjuvant was injected subcutaneouslyat days 0, 14, 28 and 56. Rabbits were bled on day 0 (pre-immunebleeding), day 38, day 66 and day 80 (final bleeding). The immunizationwas performed by CER Groupe and approved by the CER Groupe ethicalcommittee.

Serum ELISAs with GlycoDelete Proteins.

Glycosidase digestions were performed as described above. Wells ofMaxisorp microtiter plates were coated (overnight, 4° C.) with 0.25μg/mL of GM-CSF or 0.15 μg/mL of anti-CD20 in 50 μl of coating buffer(0.05 M Na2CO3, 0.05 M NaHCO3, pH 9.6) washed three times with PBS+0.1%TWEEN®, and blocked with 1% BSA in PBS with 250 mM glycine for 2 hoursat room temperature. Blocking buffer was removed and the plates weredried overnight.

Detection antibodies (anti-GM-CSF rabbit serum, final bleeding;anti-(anti-CD20) rabbit serum, final bleeding) were added in PBS+0.1%TWEEN®-20 +0.1% goat serum and incubated for 2 hours at roomtemperature.

Plates were washed four times with wash buffer before adding donkeyanti-rabbit HRP (1:2,000) (cat no. NA934, GE Healthcare) in PBS+1% BSAand incubating for 1 hour at room temperature.

The plates were washed again three times with wash buffer, upon whichthe TMB (3,3′,5,5′-tetramethylbenzidine, BD OptEIA) substrate (100 μLper well) was added and the plate was incubated at room temperature for30 minutes. Finally, 50 μL of stop solution (2 N H2SO4) was added andmeasured the absorbance at 450 nm.

The ELISA with GM-CSF was performed once with two biological replicates(two rabbits immunized; FIGS. 7E and 7F). The ELISA with anti-CD20 wasperformed once with two biological replicates (two rabbits immunized)and one of the biological replicates was then repeated with threetechnical replicates. The result of the latter experiment is shown inFIG. 14F. The data points plotted in this figure represent mean valuesfrom the three technical replicates. The error bars are s.d.

CD20 Binding by Anti-CD20.

Fc receptors on the Raji cells were blocked with anti-CD32 antibodiesIV.3 (ref. 29) (produced in-house) and AT10 (cat no. MCA1075, AbDSerotec) at 10 μg/mL and incubated with the cells for 1 hour on ice.Next, the cells were plated into a 96-well plate (105 cells per well),and the 293S or 293SGlycoDelete anti-CD20 was added in a dilution seriesstarting from 10 μg/mL. The cells were incubated for 1 hour at 4° C. andthen washed twice with PBS+2% BSA. To detect the anti-CD20, ananti-F(Ab)2 secondary antibody conjugated to DYLIGHT® 649 (cat no.109-496-097, Jackson laboratories) was added at a 1:200 dilution. Thecells were again incubated for 30 minutes at 4° C. and washed twice withPBS+2% BSA. To fix the cells, 150 μL of fixative (CellFIX, BectonDickinson) was added in each well and incubated for 1 hour at 4° C. Thesecondary antibody was detected through flow cytometry (FACS® Calibur,Becton Dickinson). The data points plotted in FIGS. 3A and 3B representmean values from three technical replicates. The error bars are s.d.This experiment was conducted twice.

FcγR Surface Plasmon Resonance Experiments.

A BIACORE® 2000 SPR biosensor (GE Healthcare) was used to assay theinteraction of FcRn with the different anti-CD20 glycoforms. Allexperiments were performed at 25° C. A CM5 chip was activated forcross-linking for 7 minutes with a solution of EDC(1-ethyl-3[3-dimethylaminopropyl]carbodiimide) and NHS(N-hydroxysuccinimide) at a flow rate of 10 μL/minute. Next, 10 μg/mL ofSTREPTAVIDIN® (Roche) in a 10 mM acetate buffer, pH 5.0, was immobilizedat the same flow rate for 7 minutes, resulting in densities ranging from1,180 to 1,280 resonance units (RU). After immobilization, the chip wasblocked by injecting 1 M of ethanolamine for 7 minutes. To finalize theimmobilization, the chip was washed three times with 20 μL of a 40 mMNaOH, 1 M NaCl buffer.

To immobilize the hFcRn on the STREPTAVIDIN® sensor surface, the pH wasbrought to 8.0 by priming with HBS-EP buffer, pH 8.0 (GE Healthcare).Biotinylated hFcRn (produced at Novlmmune)30 was diluted in HBS-EPbuffer and immobilized on the chip. Then, the system was primed withHBS-EP buffer at pH 6.0.

IgG was injected at different concentrations ranging from 67 nM to 2 nM,and diluted in HBS-EP buffer, pH 6.0. Each injection was performed for 3minutes at a flow rate of 30 μL/minute and every time in duplicate. Thedissociation was monitored for 12 minutes. HBS-EP buffer, pH 8.0, wasused for regeneration. Results were double referenced and analyzed usinga Langmuir 1:1 fitting model (BIAeval software version 4.1).

Competition ELISAs.

The wells of Maxisorp microtiter plates were coated overnight at 4° C.with coating antibody (8 μg/mL of an anti-idiotype antibody for theFcγRI ELISA; 16 μg/mL and 10 μg/mL of HZ 15C1, a humanized anti-TLR4IgG1 (NovImmune), for FcγRIIa and FcγRIIb, respectively), in 50 μl ofPBS and were then washed five times with washing buffer (PBS+0.05%TWEEN®) and blocked with 250 μL of 3% BSA in PBS per well for 1 hour at37° C. After blocking, the plates were washed five times with washingbuffer.

50 μL of anti-CD20 was added to the wells in a serial dilution indilution buffer (PBS+1% BSA) together with 50 μL of the His-tagged FcγR(FcγRI, 0.030 μg/mL; FcγRIIaR, 0.056 μg/mL; FcγRIIb, 1μg/mL (R&DSystems)). The plates were incubated for 1.5 hours at 37° C. and washedfive times with washing buffer. HRP-labeled anti-His antibody (cat no.34660, Qiagen) was added at a 1:2,000 dilution in dilution buffer andthe plates were incubated for 1 hour at 37° C. The plates were washedfive times with washing buffer before addition of 50 μL of TMBsuper-slow (Diarect) substrate. The plates were then incubated in thedark for 30 minutes. Finally, 50 μL of stop solution (2 N H2SO4) wasadded. Absorbance at 450 nm was measured with a SYNERGY® HT plate reader(Biotek).

The data points plotted in FIG. 14E (top three panels), represent meanvalues from three technical replicates. The error bars are s.e.m. Thereported differences between the 293S- and 293SGlycoDelete-producedantibodies were observed in several iterations of technical optimizationof these experiments, and the results of the fully optimized ELISAs areshown.

Biolayer Interferometry Assay.

Real-time binding of purified IgG to FcγRIIIa was evaluated usingbiolayer interferometry (BLI) on an OCTET® RED96 system (Fortebio, MenloPark, Calif.). Assays were performed at a temperature of 30° C. inkinetics buffer containing 1 mM phosphate, 15 mM NaCl, 0.002% (vol/vol)TWEEN®-20, 0.005% (wt/vol) sodium azide, 0.1 mg/mL (wt/vol) BSA, pH 7.4.FcγRIIIaV (R&D Systems, MN, USA) tagged with a hexahistidine tag wasbrought to a concentration of 1.5 μg/mL in kinetics buffer. The receptorwas captured on an anti-penta-His biosensor (Fortebio, Menlo Park,Calif.) for 10 minutes. The ligand density was 0.5 nm. Baseline signalhad stabilized after 2 minutes incubation in kinetics buffer.

A first binding assay was performed with IgG at a single concentrationof 50 μg/ml in kinetics buffer. Association and dissociation weremonitored for 5 minutes. Regeneration was performed by incubating thesensor with 10 mM glycine pH 3.0 buffer for 20 seconds, followed by 20seconds incubation in kinetics buffer. These incubations were repeatedtwice to achieve complete regeneration.

For the kinetics experiment, an F_(c)γRIIIaV-coated biosensor wasincubated with IgG at concentrations ranging from 333 nM to 19.3 nM. Atwo minute baseline situation was followed by a five-minute associationphase and a 15-minute dissociation phase in kinetics buffer.Regeneration was performed as described above. The affinity wasdetermined at equilibrium using a steady-state model. All analyses weredone using the ForteBio Data Analysis software (Fortebio, Menlo Park,Calif.).

ADCC Assay.

Peripheral blood mononuclear cells (PBMCs) were isolated from freshblood after centrifugation in a Ficoll tube (Vacutainer tube CPT, BectonDickinson). Natural killer (NK) cells were isolated from the PBMC poolusing a negative NK Cell Isolation Kit (Miltenyi Biotec). These cellswere activated overnight in growth medium (RPMI 1640+10% FBS+2 mMglutamine)+10 ng/mL IL-2.

Raji cells were seeded in a 96-well plate at 20,000 cells per well. 25μL samples of anti-CD20 antibodies were added in a 1:5 dilution series(in ADCC medium: RPMI 1640+1% BSA+2 mM glutamine+25 μg/mL gentamicin),starting with 5μg/mL. The plates were then incubated for 30 minutes at37° C. and 5% CO2. NK cells were added to the Raji cells in a ratio of1:5 (Raji/NK), and the plate was incubated at 37° C. and 5% CO2 for 4hours. Finally, the specific lysis was determined by measuring thelactate dehydrogenase (LDH) levels for each well (Cytotoxicity DetectionKit PLUS, Roche).

The data points in FIG. 14E (bottom) represent mean values from threetechnical replicates. The error bars are s.d. The reported profiles wereobserved in several iterations of technical optimization of theseexperiments and the results of the fully optimized experiment are shown.

Pharmacokinetics.

Two groups of 36 female, 8-week-old C57BL/6J mice (Charles River) wererandomly assigned to be intravenously injected with 18.5 μg (1 mg perkilogram of body weight) of either 293S or 293SGlycoDelete anti-CD20. Ateach time point (1 hour, 24 hours, 48 hours, 4 days, 7 days, 10 days, 14days, 21 days and 28 days), four mice per treatment group weresacrificed for a final bleeding, and the concentration of anti-CD20 wasdetermined with the FastELYSA human IgG kit (RD-Biotech) according tothe manufacturer's instructions. The data points shown in FIG. 14G arethe mean values (four mice) for each time point. The error bars ares.e.m. This experiment was repeated with bleedings at earlier timepoints after injection (see FIG. 17). For practical reasons, theinvestigators were not blinded to the treatment group assignment of themice. This experiment was approved by the ethical committees of GhentUniversity (Belgium) and of the Cantonal Veterinary Office of Geneva(Switzerland).

Construction of pCAGGS-s-endoT, pCAGGS-GM₂S-endoT and pCAGGS-ST-endoT.The endoT coding sequence³ without the signal sequence was amplifiedfrom a pUC19 cloning vector containing the full-size endoT codingsequence, with PCR primers PR1 and PR4 (for ST-endoT), PR2 and PR4 (forGM₂S-endoT) or PR3 and PR4 (for “endoT”). All primer sequences areprovided in Supplementary Note 2. The coding sequence for the N-terminalparts of ST6GalI⁴ (for ST-endoT) and B4GALNTI⁵ (for GM₂S-endoT) wereamplified from a human hepatoma G2 cDNA library with primers PR5, PR6and PR7, PR8, respectively. Fusion PCR reactions to generate theST-endoT, the GM₂S-endoT and endoT without signal sequence were set upusing PR5 and PR4, PR7 and PR4 and PR3 and PR4, respectively. Subsequentdigestion of the fusion PCR products ST-endoT, GM₂S-endoT and endoT withXhoI and Bsu36I and ligation into an XhoI and Bsu36I digested anddephosphorylated pCAGGS plasmid, resulted in the pCAGGS-ST-endoT andpCAGGS-GM₂S-endoT plasmids. The dsDNA signal sequence for the s-endoTconstruct was produced by annealing oligonucleotides PR9 and PR10. ThepCAGGS-endoT plasmid was digested with XhoI and KpnI. Subsequentligation of the adapter into the plasmid resulted in the pCAGGS-s-endoTplasmid.

Transfection and sample preparation. Cells were transfected as described(see online methods). Three days post-transfection with pCAGGS-s-endoT,pCAGGS-GM₂S-endoT or pCAGGS-ST-endoT, cells and supernatants wereharvested. For cell lysates, cells were collected by centrifugation at1000 rpm and washed once with PBS. Cell lysates were prepared byincubating ˜1 million cells with 500 μl RIPA buffer (150 mM sodiumchloride, 1.0% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% sodiumdodecyl sulphate and 50 mM Tris, pH 8.0) at 4° C. on a rotating platformfor 30 minutes, followed by centrifugation at 14,000 rpm for 10 minutesand discarding the insoluble material. 20 μl samples were supplementedwith 5 μl 5× SDS-PAGE loading buffer (8.3% SDS, 41.7% glycerol, 0.1%bromophenol blue, 208 mM Tris-HCl, pH 6.8 and 65 mM dithiothreitol addedfresh) and boiled for 10 minutes.

500 μl samples of cell culture supernatants were cleared bycentrifugation for 10 minutes at 14,000 rpm in a microcentrifuge,acetone precipitated by adding 2 volumes of ice cold acetone andincubated on ice for 30 minutes. Precipitated samples were centrifugedfor 10 minutes at 14,000 rpm in a microcentrifuge and the supernatantswere discarded. Pellets were dissolved by adding 80 μl of ultrapurewater and 20 μl 5× SDS-PAGE loading buffer, followed by boiling toredissolve and denature protein pellets.

Immunoblotting. 25 μl aliquots of cell lysates or supernatant sampleswere analyzed for the presence of endoT fusion proteins byimmunoblotting. Indirect detection was performed using a customgenerated rabbit polyclonal antibody against the endoT enzyme (CERgroupe, Département Santé, Marloie, Belgium). The antigen was endoTproduced in Pichia pastoris and purified previously in the lab. Thefinal antigen preparation was 1 mg/ml antigen in phosphate-bufferedsaline. The secondary antibody was an IRDye 680 goat anti-rabbit IgG(LI-COR Biosciences, Lincoln, Nebr., USA). To assess C-terminalprocessing, the same blots were probed with a mouse primary antibodydirected against the myc tag (Life Technologies, Paisley, UK) and anIRDye 800 goat anti-mouse IgG secondary antibody (LI-COR Biosciences,Lincoln, Nebr., USA).

To evaluate the in vivo de-N-glycosylation by the endoT fusion proteins,the fusion constructs were transiently transfected (transfection, seeOnline Methods) to 293SGnTI−/− cells that stably and inducibly expressedthe Flt3 receptor extracellular domain (Flt3ECD), C-terminally taggedwith a penta-His tag (cells kindly provided by Prof. Dr. S. Savvides,UGhent) or to 293SGnTI−/− cells stably and inducibly expressing5-hydroxytryptamin receptor 1D (5HT1D), C-terminally tagged with aRho1D4 tag (stable 5HT1D cell line isolation, see FIGS. 5A and 5Bmethods thereof). The producer cell lines were transfected with theendoT fusion constructs or empty plasmid and induced with 2 μg/ml tissueculture grade tetracycline and 5 mM sodium butyrate (both Sigma-Aldrich,St. Louis, Mo., USA). Supernatants (for Flt3ECD production) wereharvested 48 hours and 72 hours post-transfection/induction, or cells(for 5HT1D production) were harvested 72 hourspost-transfection/induction.

For the Flt3ECD, 20 μl aliquots of cell supernatants were run onSDS-PAGE and the processing of the Flt3 was analyzed by Westernblotting. The primary antibody was a mouse anti-penta his tag (Qiagen,Hilden, Germany) and the secondary antibody, an anti-mouse IgG-coupledto HRP (GE Healthcare Biosciences, Pittsburgh, Pa., USA).

For the 5HT1D, cells were collected by centrifugation at 1000 rpm andwashed once with PBS. Cell lysates were prepared by incubating ˜1million cells with 500 μl RIPA buffer at 4° C. on a rotating platformfor 30 minutes, followed by centrifugation at 14,000 rpm for 10 minutesand discarding the insoluble material. 20 μl samples were supplementedwith 5 μl 5× SDS-PAGE loading buffer and boiled for 10 minutes and thenloaded on a 10% SDS-PAGE gel. Western blot analysis was performed with aprimary mouse anti-Rho1D4 antibody (University of British Columbia) anda secondary anti-mouse IgG-coupled to HRP.

Early splits (#+8) of both endoT-expressing clones and 293SGnTI−/− cellswere plated in 24-well plates at 30,000 cells per well in the presenceof increasing ConA concentrations: 0-22 μg/ml. ConA was addedimmediately upon splitting. When cells in the wells containing no ConAhad grown to confluence, end points were determined microscopically. Endpoints were defined through phase contrast microscopy, as theconcentration of ConA that reduced the growth to ≤10% confluence of thewell. For assessing long term stability of endoT expression, late splitcells (#+28) were compared to early split cells (#+8).

EndoT CDS validation. To validate the presence of the CDS, genomic DNAwas prepared from ˜1 million cells of both the 293SGlycoDelete and293SGnTI−/− cell lines with the Gentra PUREGENE® Core kit A (Qiagen,Hilden, Germany), according to the manufacturer's instructions. Atouchdown PCR reaction was performed with the PHUSION® High-Fidelity DNApolymerase (New England Biolabs, Ipswich, Mass., USA) employing ˜10 nggenomic DNA for each 50 μl reaction and primers PR11 and PR12. PCRcycling was a touchdown protocol with the primer annealing temperaturelowered by 1° C. every two cycles, from 67° C. to 64° C. and held at 64°C. for 30 cycles (accounting for 36 cycles in total). PCR products wereanalyzed with a Shimadzu MULTINA™ microchip DNA/RNA electrophoresissystem, employing the DNA-500 reagent kit (Shimadzu Corporation, Kyoto,Japan) according to the manufacturer's instructions.

EndoT fusion protein validation. The expression of the ST-endoT proteinwas assessed by Western blotting. Methods are the same as described forFIGS. 1A-1D, except that the secondary antibody was an IRDye 800 Goatanti-rabbit IgG antibody (LI-COR Biosciences, Lincoln, Nebr., USA).

DSA-FACE analysis of 293S GM-CSF. N-linked oligosaccharides wereprepared from purified proteins upon blotting to PVDF membrane in thewells of 96-well plate membrane plates, and were analyzed by capillaryelectrophoresis with laser-induced fluorescence detection (CE-LIF) usingan ABI 3130 capillary DNA sequencer as described previously.⁶

TABLE 4 (Data underlying FIG. 1C) Cells/well hours Well 1 Well 2 Well 3Avg St Dev Cells/well St dev 293SGlycoDelete 0 27 23 21 23.67 3.06157778 20367 24 36 37 26 33.00 6.08 220000 40552 48 53 42 39 44.67 7.37297778 49141 72 150 194 208 184.00 30.27 1226667 201770 96 270 282 234262.00 24.98 1746667 166533 120 468 510 378 452.00 67.44 3013333 449592144 492 519 465 492.00 27.00 3280000 180000 168 549 510 504 521.00 24.433473333 162891 192 512 476 532 506.67 28.38 3377778 189189 293SGnTl−/− 029 27 23 26.33 3.06 175556 20367 24 55 58 45 52.67 6.81 351111 45379 4851 61 72 61.33 10.50 408889 70026 72 199 176 168 181.00 16.09 1206667107290 96 266 268 210 248.00 32.92 1653333 219494 120 360 404 402 388.6724.85 2591111 165641 144 450 447 468 455.00 11.36 3033333 75719 168 501444 471 472.00 28.51 3146667 190088 192 516 464 496 492.00 26.23 3280000174865

TABLE 5 (data underlying FIG. 7D) ng/mL 293S 293 GlycoDelete E. Coli 540.686 0.709 0.702 0.665 0.637 0.731 0.612 0.661 0.671 18 0.687 0.6570.648 0.691 0.658 0.724 0.627 0.705 0.642 6 0.593 0.665 0.619 0.63 0.670.693 0.632 0.704 0.601 2 0.588 0.646 0.593 0.615 0.587 0.719 0.4950.577 0.525 0.6667 0.548 0.605 0.555 0.568 0.601 0.674 0.298 0.37 0.3080.2222 0.463 0.522 0.49 0.528 0.557 0.618 0.138 0.185 0.143 0.0741 0.2660.384 0.321 0.365 0.446 0.446 0.042 0.074 0.053 0.0247 0.133 0.202 0.170.248 0.27 0.314 −0.021 0.015 −0.026 0.008 0.048 0.076 0.068 0.142 0.1320.156 −0.056 −0.042 −0.069 0 −0.07 −0.088 −0.082 −0.057 −0.102 −0.059−0.081 −0.084 −0.101

TABLE 6 (data underlying FIG. 7E) eT eT sialidase + eT eT not nosialidase + et eT not no galactosidase/ sialidase/ treated/ protein/galactosidase/ sialidase/ treated/ protein/ Antibody Antibody R2 R2 R2R2 R1 R1 R1 R1 dilution dilution 3.5 3.5 3.5 0.276 3.5 3.5 3.5 0.396 1 13.5 3.5 3.5 0.189 3.5 3.5 3.5 0.197 2 2 3.5 3.5 3.5 0.16 3.5 3.5 3.50.187 4 4 3.5 3.5 3.5 0.195 3.5 3.5 3.5 0.218 8 8 3.5 3.5 3.5 0.1383.058 3.161 3.015 0.151 16 16 3.225 3.279 3.15 0.126 2.307 2.326 2.1980.16 32 32 2.789 2.754 2.695 0.144 1.564 1.554 1.462 0.166 64 64 1.9031.865 1.792 0.123 0.907 0.924 0.862 0.098 128 128 1.192 1.201 1.13 0.1470.57 0.538 0.533 0.107 256 256 0.723 0.746 0.69 0.106 0.332 0.353 0.3630.08 512 512 0.439 0.449 0.419 0.125 0.228 0.218 0.199 0.077 1024 10240.106 0.102 0.115 0.099 0.128 0.098 0.102 0.075 2048 2048

TABLE 7 (data underlying FIG. 14C Antibody concentration Anti-CD20 μg/mLAnti-CD20 293 293 GlycoDelete 10 2637.7 2563.4 2582.8 2633.8 2599.32539.1 2.5 2145.8 2177.3 1924.2 2223.5 2130.6 2250.8 0.625 1501.8 1516.71548.3 1434.7 1511.1 1554.9 0.15625 673.3 673.1 644 602.4 662.6 667.10.039063 240.9 234 210.2 206.7 216.2 248.7 0.009766 76.4 79.7 72.2 71.568.2 75.9 0.002441 28.6 27.5 23.2 26.8 24.6 26.9 0.00061 14.1 12.3 11.314 10 19

TABLE 8 (data underlying FIG. 14E FcγRI Ab Concentration μg/mlGlycoDelete Anti-CD20 293 Anti-CD20 50 0.315 0.324 0.317 0.085 0.0910.084 10 1.163 1.153 1.143 0.143 0.143 0.141 2 2.418 2.324 2.324 0.3880.379 0.377 0.4 3.079 2.886 2.961 1.138 1.105 1.102 0.08 3.215 3.1653.257 2.243 2.198 2.332 0.016 3.389 3.339 3.387 3.049 3.036 3.156 0.00323.379 3.443 3.344 3.394 3.397 3.437 0.0006 3.474 3.589 3.506 3.428 3.5983.602 FcγRIIa Antibody Concentration Anti-CD20 μg/ml 293 GlycoDeleteAnti-CD20 293 2.69897 1.559 2.638 2.573 0.684 0.732 0.671 2.22185 2.4082.7 2.761 1.502 1.644 1.682 1.744731 2.182 2.905 2.918 2.113 2.463 2.3591.267617 2.529 3.021 3.014 2.433 2.907 2.725 0.790496 2.389 2.967 3.1912.564 2.907 2.981 0.313445 2.521 3.019 3.103 2.569 2.965 2.932 −0.163682.441 2.944 3.068 2.687 3.023 3.099 −0.64016 2.438 3.058 3.031 2.6052.885 2.98 (data underlying FIG. 14F) FcγRIIb Antibody ConcentrationAnti-CD20 293 μg/ml GlycoDelete Anti-CD20 293 2300 0.962 0.84 766.671.476 1.356 255.56 1.56 1.62 85.19 1.769 1.678 28.4 1.739 1.73 9.471.871 1.814 3.16 1.899 1.827 3000 0.284 0.274 1000 0.641 0.629 333.331.061 1.098 111.11 1.517 1.49 37.04 1.662 1.545 12.35 1.991 1.874 4.122.268 1.918 ADCC Antibody Concentration Anti-CD20 293 μg/ml GlycoDeleteAnti-CD20 293 2.3 52.44 50.35 0.46 46.6 48.82 0.092 42.04 45.15 0.018433.85 32.4 0.00368 16.5 15.22 0.000736 4.52 4.43 0.000147 0.26 −1.112.94E−05 1.66 −0.17 3 52.14 56.15 0.6 53.29 49.37 0.12 51.84 51.42 0.02451.76 49.07 0.0048 43.7 44.64 0.00096 21.62 21.36 0.000192 4.26 8.443.84E−05 2.26 1.45

TABLE 9 (data underlying FIG. 14F) 293SGlycoDelete a-CD20 +galactosidase + 293SGlycoDelete 293SGlycoDelete Antibody sialidasea-CD20 + sialidase a-CD20 No protein dilution 3.5 3.43 3.5 3.5 3.5 3.53.5 3.428 3.5 0.459 0.458 0.484 50 3.5 3.5 3.5 3.441 3.5 3.5 3.5 3.5 3.50.179 0.161 0.162 150 3.231 3.147 3.212 3.16 3.398 3.335 3.252 3.2833.25 0.077 0.081 0.019 450 2.778 2.628 2.851 2.815 2.879 2.864 2.7982.689 2.906 0.054 0.068 0.025 1350 1.902 1.756 1.914 1.992 2.159 2.3871.982 1.968 2.043 0.034 0.043 0.016 4050 1.048 0.931 1.024 1.09 1.2811.308 1.07 1.045 1.188 0.02 0.049 0.013 12150 0.477 0.494 0.456 0.5240.48 0.619 0.5 0.513 0.278 0.023 0.046 0.061 36450 0.225 0.211 0.1980.232 0.215 0.274 0.216 0.21 0.207 0.02 0.037 0.002 109350 0.121 0.1030.085 0.116 0.1 0.186 0.109 0.138 0.081 0.02 0.021 0.001 328050 0.0830.074 0.043 0.07 0.06 0.051 0.073 0.053 0.035 0.018 0.03 0.005 984150

TABLE 10 (data underlying FIG. 14G) Time (h) Anti-CD20 293s Anti-CD20293GlycoDelete 1 16.1974 13.17261 17.00356 11.66813 28.8332 29.387733.474 36.6924 24 7.4006 9.311962 8.466237 6.55145 14.78954 16.358914.10662 13.68416 48 7.21618 7.227962 6.746963 7.31205 19.8775 13.9302315.7465 17.25974 96 6.76923 8.425325 6.122825 7.1901 12.1798 14.3152217.8752 14.34155 168 5.17352 7.374187 5.897713 6.8257 11.631 14.2136611.92079 11.60133 240 4.16867 3.625075 5.712138 6.59155 11.5113511.86461 10.81969 11.3477 336 3.88537 4.81935 4.346875 2.606795 7.23968710.82754 7.92285 7.9431 504 3.13270 2.52692 2.639255 2.566235 5.3577756.811675 5.078675 5.5248 672 1.59795 1.916417 1.99827 1.65403 3.3496953.54475 3.661225 3.075095

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Additional References for Methods Section

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1-15. (canceled)
 16. A plurality of Fc-containing molecules: wherein theFc-containing molecules are glycosylated on N297 of the Fc part, whereinthe glycosylation on N297 of the Fc part consists of one or more glycansselected from the group consisting of a trisaccharide structureNeu5Ac-Hex-HexNAc, a disaccharide structure Hex-HexNAc, and amonosaccharide structure HexNAc; and wherein at least one of theplurality of Fc-containing molecules comprises a glycan that is not amonosaccharide structure HexNAc.
 17. The plurality of Fc-containingmolecules of claim 16, wherein the Fc-containing molecules of theplurality are identical.
 18. The plurality of Fc-containing molecules ofclaim 16, wherein the trisaccharide structure isNeu5Ac-α-2,3-Gal-β-1,4-GlcNAc, the disaccharide structure isGal-β-1,4-GlcNAc, and the monosaccharide structure is GlcNAc.
 19. Theplurality of Fc-containing molecules of claim 16, wherein theFc-containing molecules are antibodies.
 20. The plurality ofFc-containing molecules of claim 17, wherein the Fc-containing moleculesare antibodies.
 21. The plurality of Fc-containing molecules of claim18, wherein the Fc-containing molecules are antibodies.
 22. A medicamentcomprising: the plurality of Fc-containing molecules of claim
 16. 23. Amedicament comprising: the plurality of Fc-containing molecules of claim17.
 24. A medicament comprising: the plurality of Fc-containingmolecules of claim
 18. 25. A medicament comprising: the plurality ofFc-containing molecules of claim
 19. 26. A medicament comprising: theplurality of Fc-containing molecules of claim
 20. 27. A medicamentcomprising: the plurality of Fc-containing molecules of claim
 21. 28. Amethod of increasing circulation time of an Fc-containing molecule,without altering antigen binding, in a subject in need thereof, themethod comprising: administering the plurality of Fc-containingmolecules of claim 16 to the subject.
 29. An antibody comprisingglycosylation on N297 of the Fc part thereof wherein the glycosylationon N297 comprises: a trisaccharide structure that isNeu5Ac-α-2,3-Gal-β-1,4-GlcNAc; a disaccharide structure that isGal-β-1,4-GlcNAc; and a monosaccharide structure that is GlcNAc.
 30. Theantibody of claim 29, which is an IgG.